Anti-human papillomavirus (hpv) antigen-binding proteins and methods of use thereof

ABSTRACT

The present invention provides antigen-binding proteins that specifically bind to an HLA-displayed human papillomavirus (HPV) peptide, and therapeutic and diagnostic methods of using those binding proteins.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/019,703, filed on Jun. 27, 2018, which claims priority to U.S.Provisional Application No. 62/525,937, filed on Jun. 28, 2017, theentire contents of which are expressly incorporated by reference hereinin their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Aug. 7, 2020, is named118003_28903_SL.txt and is 253,645 bytes in size.

FIELD OF THE INVENTION

The present invention is related to antigen-binding proteins thatspecifically bind to an HLA-displayed human papillomavirus (HPV)peptide, and therapeutic and diagnostic methods of using those bindingproteins.

BACKGROUND OF THE INVENTION

Human papillomavirus (HPV) is a group of small, non-enveloped DNAviruses that are extremely common worldwide. HPV is mainly transmittedthrough sexual contact and most people are infected with HPV shortlyafter the onset of sexual activity.

There are more than 170 types of HPV, some of which can cause warts orbenign papillomas, and others, at least 13 of which, cause cancer (alsoknown as high risk type HPVs), including cervical cancer, anogenitalcancers (cancers of the anus, penis, vagina and vulva), head/neckcancers, and oropharynx cancers, including the back of the throat, thebase of the tongue, and tonsils. Indeed, HPV is present in 20-40% of allhead and neck squamous cell carcinomas (HNSCC) and in 100% of cervicalcancers.

Cervical cancer is the second most common cancer in women living in lessdeveloped regions with an estimated 445,000 new cases in 2012 (84% ofthe new cases worldwide). In 2012, approximately 270,000 women died fromcervical cancer; more than 85% of these deaths occurring in low- andmiddle-income countries.

Two HPV types (16 and 18) cause approximately 70% of all cervicalcancers and precancerous cervical lesions. Cancer development uponpersistent infection with a high risk HPV subtype, such as HPV 16 or 18,is mainly attributable to the expression of two viral oncoproteins, E6and E7, which are continuously expressed in lesions and presented on thecell surface by MHC class I, but are not expressed in normal cells. E6and E7 promote genomic instability and cellular transformation bydegrading the tumor suppressors p53 and Rb in a proteasome-dependentmanner. Tumors arise several years after the initial cellularimmortalizing events and the continuous expression of E6 and E7 isrequired for maintenance of the transformed phenotype, and prevention ofcell growth arrest and/or apoptosis (McLaughlin-Drubin M. E. & MiingerK., Virology (2009) 384:335-344).

Although vaccines targeting the HPV L1 and L2 major capsid proteins ofHPV-6, -11, -16 and -18 subtypes have been developed to preventinfection, such vaccines cannot treat subjects having establishedlesions. Thus, the treatment of subjects having cervical cancer remainsthe use of traditional approaches which are highly invasive and morbid,such as surgery, radiotherapy, and chemotherapy. Furthermore, althoughsuch treatments may provide benefit for subjects having early stagecervical cancer, they are of limited value to patients with advanced orrecurrent cervical cancer.

Accordingly, there is an unmet need in the art for new therapeuticstrategies to target HPV with high specificity and to treat cervicalcancer and other cancers caused by HPV.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antigen-binding proteins thatspecifically bind to a conformational epitope of an HLA-displayed humanpapillomavirus (HPV) 16 E7 peptide (HLA-A2:HPV16E7). The antigen-bindingproteins of the present invention bind with a high degree of specificityto HLA-displayed HPV16E7 and do not bind to HLA-displayed peptides thatdiffer by 1, 2, 3, 4, 5 or more amino acids. The antigen-bindingproteins of the invention allow for specific targeting of HPV16E7peptide-presenting cells (i.e., cells presenting on their surface anHPV16E7 peptide bound to an MHC molecule, e.g., HLA-A2), such as cancercells expressing HPV16E7 and, in some embodiments, stimulating T cellactivation, e.g., to stimulate T cell-mediated killing of such cells.Furthermore, when fused to a detectable moiety, the antigen-bindingproteins of the present invention allow for diagnosis and prognosis ofHPV16E7-positive diseases or disorders with high sensitivity to changesin the number and distribution of HPV16E7 peptide-presenting cells, amore relevant measure of disease progression than circulating HPV16E7levels.

The antigen-binding proteins of the invention may be antibodies, such asfull-length (for example, an IgG1 or IgG4 antibody) antibodies, or maycomprise only an antigen-binding portion of an antibody (for example, aFab, F(ab)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933). In some embodiments, theantigen-binding proteins of the invention may be antibodies, orantigen-binding fragments thereof. In certain embodiments, theantigen-binding proteins may be bispecific.

In a first aspect, the present invention provides isolated recombinantantigen-binding proteins that bind specifically to a conformationalepitope of an HLA-displayed human papillomavirus (HPV) 16 E7 peptide,such as a HLA-displayed peptide comprising amino acid residues 11-19 or82-90 of HPV16E7. In certain embodiments, the antigen-binding proteinsare antibodies. In some embodiments, the antibodies are fully human.

Exemplary anti-HLA-A2:HPV16E7 antigen-binding proteins of the presentinvention are listed in Tables 1 and 2 herein. Table 1 sets forth theamino acid sequence identifiers of the heavy chain variable regions(HCVRs), light chain variable regions (LCVRs), heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3), and lightchain complementarity determining regions (LCDR1, LCDR2 and LCDR3) ofthe exemplary anti-HLA-A2:HPV16E7 antibodies. Table 2 sets forth thenucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-HLA-A2:HPV16E7antibodies.

The present invention provides antigen-binding proteins comprising anHCVR comprising an amino acid sequence selected from any of the HCVRamino acid sequences listed in Table 1, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antigen-binding proteins comprisingan LCVR comprising an amino acid sequence selected from any of the LCVRamino acid sequences listed in Table 1, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antigen-binding proteins comprisingan HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising anyof the HCVR amino acid sequences listed in Table 1 paired with any ofthe LCVR amino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antigen-binding proteinscomprising an HCVR/LCVR amino acid sequence pair contained within any ofthe exemplary anti-HLA-A2:HPV16E7 antigen-binding proteins listed inTable 1. In certain embodiments, the HCVR/LCVR amino acid sequence pairis selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290,298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418,426/434, 442/450, 458/466, 474/482, 490/498, 506/514, and 522/530. Incertain embodiments, the HCVR/LCVR amino acid sequence pair is selectedfrom one of SEQ ID NOs: 2/10 (e.g., H4sH17364N), 34/42 (e.g.,H4sH17670P), 82/90 (e.g., H4sH17675P), 194/202 (e.g., H4sH17930N2),282/290 (e.g., H4sH21064P), and 506/514 (e.g., H4sH17363N).

In certain embodiments, the present invention providesanti-HLA-A2:HPV16E7 antigen-binding proteins comprising a HCVR and aLCVR, said HCVR comprising an amino acid sequence listed in Table 1having no more than five amino acid substitutions, and said LCVRcomprising an amino acid sequence listed in Table 1 having no more thanfive amino acid substitutions. For example, the present inventionprovides anti-HLA-A2:HPV16E7 antigen-binding proteins comprising a HCVRand a LCVR, said HCVR comprising an amino acid sequence of SEQ ID NO:194 having no more than five amino acid substitutions, and said LCVRcomprising an amino acid sequence of SEQ ID NO: 202 having no more thanfive amino acid substitutions. In another exemplary embodiment, thepresent invention provides anti-HLA-A2:HPV16E7 antigen-binding proteinscomprising a HCVR and a LCVR, said HCVR comprising an amino acidsequence of SEQ ID NO: 194 having at least one amino acid substitution,and said LCVR comprising an amino acid sequence of SEQ ID NO: 202 havingat least one amino acid substitution.

The present invention also provides antigen-binding proteins comprisinga heavy chain CDR1 (HCDR1) comprising an amino acid sequence selectedfrom any of the HCDR1 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga heavy chain CDR2 (HCDR2) comprising an amino acid sequence selectedfrom any of the HCDR2 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga heavy chain CDR3 (HCDR3) comprising an amino acid sequence selectedfrom any of the HCDR3 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga light chain CDR1 (LCDR1) comprising an amino acid sequence selectedfrom any of the LCDR1 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga light chain CDR2 (LCDR2) comprising an amino acid sequence selectedfrom any of the LCDR2 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga light chain CDR3 (LCDR3) comprising an amino acid sequence selectedfrom any of the LCDR3 amino acid sequences listed in Table 1 or asubstantially similar sequence thereof having at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity.

The present invention also provides antigen-binding proteins comprisinga HCDR3 and a LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprisingany of the HCDR3 amino acid sequences listed in Table 1 paired with anyof the LCDR3 amino acid sequences listed in Table 1. According tocertain embodiments, the present invention provides antigen-bindingproteins, comprising an HCDR3/LCDR3 amino acid sequence pair containedwithin any of the exemplary anti-HLA-A2:HPV16E7 antigen-binding proteinslisted in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acidsequence pair is selected from the group consisting of SEQ ID NOs: 8/16(e.g., H4sH17364N), 40/48 (e.g., H4sH17670P), 88/96 (e.g., H4sH17675P),200/208 (e.g., H4sH17930N2), 288/296 (e.g., H4sH21064P), and 512/520(e.g., H4sH17363N).

The present invention also provides antigen-binding proteins comprisinga HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acidsequence differing from an amino acid sequence listed in Table 1 by 1amino acid, HCDR2 comprising an amino acid sequence differing from anamino acid sequence listed in Table 1 by 1 amino acid, and HCDR3comprising an amino acid sequence differing from an amino acid sequencelisted in Table 1 by 1 amino acid. In certain embodiments, the presentinvention provides antigen-binding proteins comprising a HCVR and aLCVR, said LCVR comprising LCDR1 comprising an amino acid sequencediffering from an amino acid sequence listed in Table 1 by 1 amino acid,LCDR2 comprising an amino acid sequence differing from an amino acidsequence listed in Table 1 by 1 amino acid, and LCDR3 comprising anamino acid sequence differing from an amino acid sequence listed inTable 1 by 1 amino acid. For example, the present invention providesanti-HLA-A2:HPV16E7 antigen-binding proteins comprising a HCVR and aLCVR, said HCVR comprising HCDR1 comprising an amino acid sequence ofSEQ ID NO: 196 or an amino acid sequence differing from SEQ ID NO: 196by 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO:198 or an amino acid sequence differing from SEQ ID NO: 198 by 1 aminoacid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 200 oran amino acid sequence differing from SEQ ID NO: 200 by 1 amino acid. Inanother exemplary embodiment, the present invention providesantigen-binding proteins comprising a HCVR and a LCVR, said LCVRcomprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 204 oran amino acid sequence differing from SEQ ID NO: 204 by 1 amino acid,LCDR2 comprising an amino acid sequence of SEQ ID NO: 206 or an aminoacid sequence differing from SEQ ID NO: 206 by 1 amino acid, and LCDR3comprising an amino acid sequence of SEQ ID NO: 208 or an amino acidsequence differing from SEQ ID NO: 208 by 1 amino acid.

The present invention also provides antigen-binding proteins comprisinga set of six CDRs HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained withinany of the exemplary antigen-binding proteins listed in Table 1. Incertain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequence set is selected from the group consisting of SEQ ID NOs:4-6-8-12-14-16 (e.g., H4sH17364N), 36-38-40-44-46-48 (e.g., H4sH17670P),84-86-88-92-94-96 (e.g., H4sH17675P), 196-198-200-204-206-208 (e.g.,H4sH17930N2), 284-286-288-292-294-296 (e.g., H4sH21064P), and508-510-512-516-518-520 (e.g., H4sH17363N).

In a related embodiment, the present invention provides antigen-bindingproteins comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary antigen-bindingproteins listed in Table 1. For example, the present invention includesantigen-binding proteins comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/10 (e.g., H4sH17364N), 34/42 (e.g.,H4sH17670P), 82/90 (e.g., H4sH17675P), 194/202 (e.g., H4sH17930N2),282/290 (e.g., H4sH21064P), and 506/514 (e.g., H4sH17363N).

Methods and techniques for identifying CDRs within HCVR and LCVR aminoacid sequences are well known in the art and can be used to identifyCDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antigen-binding protein.

The present invention includes anti-HLA-A2:HPV16E7 antigen-bindingproteins having a modified glycosylation pattern. In some embodiments,modification to remove undesirable glycosylation sites may be useful, oran antibody lacking a fucose moiety present on the oligosaccharidechain, for example, to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In certain embodiments, the antigen-binding proteins of the inventionare monoclonal antibodies comprising a HCVR and a LCVR amino acidsequence pair (HCVR/LCVR) comprising any of the HCVR amino acidsequences listed in Table 1 paired with any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the monoclonalantibodies comprise a Fc domain of an isotype selected from the groupconsisting of IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM and avariant thereof.

The present invention provides antigen-binding proteins, orantigen-binding fragments thereof, comprising a heavy chain comprisingan amino acid sequence selected from any of the HC amino acid sequenceslisted in Table 3, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides antigen-binding proteins, orantigen-binding fragments thereof, comprising a light chain comprisingan amino acid sequence selected from any of the LC amino acid sequenceslisted in Table 3, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides antigen-binding proteins, orantigen-binding fragments thereof, comprising a HC and a LC amino acidsequence pair (HC/LC) comprising any of the HC amino acid sequenceslisted in Table 3 paired with any of the LC amino acid sequences listedin Table 3. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHC/LC amino acid sequence pair contained within any of the exemplaryanti-PD-1 antibodies listed in Table 3. In certain embodiments, theHC/LC amino acid sequence pair is selected from the group consisting ofSEQ ID NOs: 578/579, 580/581, 582/583, 584/585, 586/587, 588/589,590/591, and 592/593.

In one aspect, the present invention provides antigen-binding proteinsor antigen-binding fragments thereof that bind to a HLA-peptide complexwherein the antigen-binding protein or antigen-binding fragment thereofcontacts at least 60%, at least 70%, at least 80% or at least 90% of theamino acid residues of the peptide that is comprised in the HLA-peptidecomplex. In certain embodiments, the antigen-binding protein orantigen-binding fragment thereof “covers” or contacts all of the aminoacid residues of the peptide comprised in the HLA-peptide complex. Incertain embodiments, the antigen-binding protein or antigen-bindingfragment thereof binds to a HLA-peptide complex with high affinity andspecificity, wherein the antigen-binding protein or antigen-bindingfragment thereof contacts the entire length of the displayed peptide.“Contact”, as used herein includes direct or water-mediated hydrogenbonds, charge-charge interactions, or hydrophobic/van der Waalsinteractions. In one embodiment, the antigen-binding protein orantigen-binding fragment thereof binds to HLA-A2-HPV16E7 11-19 peptidecomplex wherein the antigen-binding protein binds to at least 6 of 10amino acid residues of peptide 11-19 (SEQ ID NO: 538) and to HLA-A2 suchthat it covers the HLA-A2-peptide complex completely. In certainembodiments, the antigen-binding protein or antigen-binding fragmentthereof comprises the CDRs of a HCVR and the CDRs of a LCVR, wherein theHCVR and LCVR each has an amino acid sequence selected from the HCVR andLCVR sequences listed in Table 1. In one embodiment, the antigen-bindingprotein is fully human. In certain embodiments, the fully humanantigen-binding proteins are not obtained using phage display methodsand technologies. In one embodiment, the antigen-binding proteinscomprise a light chain variable region of the IGKV1-39 sub-type.

In certain embodiments, the present invention provides antigen-bindingproteins or antigen-binding fragments thereof that bind toHLA-A2:HPV16E7 11-19 peptide, wherein the antigen-binding protein bindsto one or more amino acids of SEQ ID NO: 538. In one embodiment, theantigen-binding protein binds to at least 6 amino acids of SEQ ID NO:538. In one embodiment, the antigen-binding protein binds to one or moreamino acids selected from the group consisting of Y11, D14, L15, P 17and E18 of SEQ ID NO: 538.

In certain embodiments, the present invention provides antigen-bindingprotein that binds specifically to a conformational epitope of an HLA-A2presented human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide),wherein the conformational epitope comprises one or more amino acids ofSEQ ID NO: 538. In the certain embodiments, the conformational epitopecomprises one or more amino acids selected from the group consisting ofY11, D14, L15, P 17 and E18 of SEQ ID NO: 538.

The present invention also provides for antigen-binding proteins thatcompete for specific binding to HLA-A2:HPV16E7 with an antigen-bindingprotein comprising the CDRs of a HCVR and the CDRs of a LCVR, whereinthe HCVR and LCVR each has an amino acid sequence selected from the HCVRand LCVR sequences listed in Table 1.

The present invention also provides antigen-binding proteins thatcross-compete for binding to HLA-A2:HPV16E7 with a referenceantigen-binding protein comprising the CDRs of a HCVR and the CDRs of aLCVR, wherein the HCVR and LCVR each has an amino acid sequence selectedfrom the HCVR and LCVR sequences listed in Table 1.

The present invention also provides antigen-binding proteins that bindto the same epitope as a reference antigen-binding protein comprisingthe CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVReach has an amino acid sequence selected from the HCVR and LCVRsequences listed in Table 1. In certain embodiments, the presentinvention provides antigen-binding proteins that bind to the sameepitope as a reference antigen-binding protein comprising the CDRs of aHCVR and the CDRs of a LCVR, wherein the HCVR is selected from the groupconsisting of SEQ ID NOs: 2, 34, 82, 194, 282 and 504, and the LCVR isselected from the group consisting of SEQ ID Nos: 10, 42, 90, 202, 290and 514.

In one embodiment, the invention provides a recombinant isolatedantigen-binding protein that binds specifically to a conformationalepitope of an HLA-A2 presented human papillomavirus (HPV) 16 E7 peptide(HPV16E7 peptide), wherein the antigen-binding protein has a propertyselected from the group consisting of: (a) binds monomericHLA-A2:HPV16E7 11-19 peptide with a binding dissociation equilibriumconstant (K_(D)) of less than about 20 nM as measured in a surfaceplasmon resonance assay at 25° C.; (b) binds monomeric HLA-A2:HPV16E782-90 peptide with a binding dissociation equilibrium constant (K_(D))of less than about 25 nM as measured in a surface plasmon resonanceassay at 25° C.; (c) binds to HLA-A2:HPV16E7 11-19 peptide expressingcells with an EC₅₀ less than about 6 nM and does not bind to cellsexpressing predicted off-target peptides as determined by luminescenceassay; (d) binds to HLA-A2:HPV16E7 82-90 peptide expressing cells withan EC₅₀ less than about 1 nM and do not substantially bind to cellsexpressing predicted off-target peptides as determined by luminescenceassay; (e) binds to HLA-A2:HPV16E7 11-19 peptide expressing cells withan EC₅₀ less than about 30 nM as determined by flow cytometry assay; (f)binds to HLA-A2:HPV16E7 82-90 peptide expressing cells with an EC₅₀ lessthan about 75 nM as determined by flow cytometry assay; and (g) theconformational epitope comprises one or more amino acids of SEQ ID NO:538. As disclosed elsewhere herein, an “off-target peptide” refers to apeptide that differs by 1, 2, 3, 4, 5 or more amino acids from a targetpeptide (e.g., HPV16 E7 11-19 peptide).

In a second aspect, the present invention provides nucleic acidmolecules encoding anti-HLA-A2:HPV16E7 antigen-binding proteins. Forexample, the present invention provides nucleic acid molecules encodingany of the HCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-HLA-A2:HPV16E7antigen-binding proteins listed in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-HLA-A2:HPV16E7antigen-binding proteins listed in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity thereto. In certain embodiments according to thisaspect of the invention, the nucleic acid molecule encodes an HCVR andLCVR, wherein the HCVR and LCVR are both derived from the sameanti-HLA-A2:HPV16E7 antigen-binding protein listed in Table 1.

The present invention provides nucleic acid molecules encoding any ofthe heavy chain amino acid sequences listed in Table 3. The presentinvention also provides nucleic acid molecules encoding any of the lightchain amino acid sequences listed in Table 3.

The present invention also provides nucleic acid molecules encoding bothheavy chain (HC) and a light chain (LC), wherein the HC comprises anamino acid sequence of any of the HC amino acid sequences listed inTable 3, and wherein the LC comprises an amino acid sequence of any ofthe LC amino acid sequences listed in Table 3.

In a related aspect, the present invention provides recombinantexpression vectors capable of expressing a polypeptide comprising aheavy and/or or light chain variable region of an anti-HLA-A2:HPV16E7antigen-binding protein. For example, the present invention includesrecombinant expression vectors comprising any of the nucleic acidmolecules mentioned above, i.e., nucleic acid molecules encoding any ofthe HCVR, LCVR, and/or CDR sequences as set forth in Table 1. Thepresent invention also provides recombinant expression vectors capableof expressing a polypeptide comprising a heavy and/or light chain of ananti-HLA-A2:HPV16E7 antigen-binding protein. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the heavy chain or light chain sequences as set forth inTable 2. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antigen-binding proteins by culturing the hostcells under conditions permitting production of the antigen-bindingproteins, and recovering the antigen-binding proteins so produced.

In a third aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of arecombinant isolated antigen-binding protein that binds specifically toa conformational epitope of an HLA-A2 presented HPV16E7 peptide (e.g., apeptide comprising amino acid residues 11-19 or 82-90 of HPV16E7), and apharmaceutically acceptable carrier. In a related aspect, the inventionfeatures a composition which is a combination of an anti-HLA-A2:HPV16E7antigen-binding protein and a second therapeutic agent. In oneembodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-HLA-A2:HPV16E7 antigen-bindingprotein. Exemplary agents that may be advantageously combined with ananti-HLA-A2:HPV16E7 antigen-binding protein include, without limitation,other agents that bind and/or modulate HPV replication or infection(including other antibodies or antigen-binding fragments thereof, etc.)and/or agents which modulate immune cell activation. Additionaltherapies that can be used in combination with the anti-HLA-A2:HPV16E7antigen-binding proteins of the present invention are disclosedelsewhere herein.

In a fourth aspect, the invention provides methods to treat a subjecthaving an HPV-associated disease or disorder, such as anHPV16E7-positive cancer. The methods include administering atherapeutically effective amount of an anti-HLA-A2:HPV16E7antigen-binding protein of the invention or a pharmaceutical compositionof the invention to the subject in need thereof. The disorder treated isany disease or condition which is improved, ameliorated, inhibited orprevented by the antigen-binding proteins and compositions providedherein. In certain embodiments, the antigen-binding protein (orpharmaceutical composition) of the invention is administered incombination with a second therapeutic agent to the subject in needthereof. The second therapeutic agent may be selected from the groupconsisting of an antibody to a T cell co-inhibitor, an antibody to atumor cell antigen, an antibody to a T cell receptor, an antibody to anepitope on a virally infected cell, a cytotoxic agent, an anti-cancerdrug, an anti-viral drug, an anti-inflammatory drug (e.g.,corticosteroids), chemotherapeutic agent, surgery, radiation therapy, animmunosuppressant and any other drug or therapy known in the art. Incertain embodiments, the second therapeutic agent may be an agent thathelps to counteract or reduce any possible side effect(s) associatedwith antigen-binding protein of the invention, if such side effect(s)should occur.

In certain embodiments, the present invention provides methods forsuppressing growth of a HPV-associated cancer. For example, the presentinvention provides methods to suppress tumor growth due to a primarytumor or a metastatic tumor in a subject. In certain embodiments, thepresent invention provides methods to enhance survival (e.g.,progression-free survival or overall survival) of a subject with aHPV-associated cancer. Examples of cancer include, but are not limitedto, squamous cell carcinomas, such as squamous cell carcinoma of headand neck, cervical cancer, anogenital cancer, oropharyngeal cancer.

In certain embodiments, the present invention provides methods forinhibiting or suppressing growth of established tumors. The methodscomprise administering to a subject in need thereof a pharmaceuticalcomposition comprising a therapeutically effective amount of anantigen-binding protein of the present invention. In certainembodiments, the antigen-binding protein is administered in combinationwith a second therapeutic agent.

The antigen-binding protein, e.g., antibody, or antigen-binding fragmentthereof, may be administered subcutaneously, intravenously,intradermally, intraperitoneally, orally, intramuscularly, orintracranially. The antigen-binding protein, e.g., antibody orantigen-binding fragment thereof, may be administered at a dose of about0.1 mg/kg of body weight to about 100 mg/kg of body weight of thesubject.

In a fifth aspect, the present invention provides an isolated nucleicacid molecule encoding a chimeric antigen receptor (CAR). The CAR mayinclude an extracellular binding domain that specifically binds to aconformational epitope of an HLA-A2 presented human papillomavirus (HPV)16 E7 peptide (HPV16E7 peptide), e.g., amino acid residues 11-19 or82-90 of HPV16E7, a transmembrane domain, and an intracellular signalingdomain. In one embodiment, the extracellular binding domain is ananti-HLA-A2:HPV16E7 antigen-binding protein or an antigen-bindingfragment thereof. Exemplary anti-HLA-A2:HPV16E7 antigen-binding proteinsof the present invention are any of the antigen-binding proteinsdescribed herein.

For example, in certain embodiments, the antigen-binding proteinsuitable for use in the CARs of the invention comprises three heavychain complementarity determining regions (CDRs) (HCDR1, HCDR2 andHCDR3) contained within any one of the heavy chain variable region(HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1,LCDR2 and LCDR3) contained within any one of the light chain variableregion (LCVR) sequences listed in Table 1.

In other embodiments, the antigen-binding protein suitable for use inthe CARs of the invention comprises a HCVR having an amino acid sequenceselected from the group consisting of HCVR sequences listed in Table 1;and/or a LCVR having an amino acid sequence selected from the groupconsisting of LCVR sequences listed in Table 1.

In some embodiments, the antigen-binding protein suitable for use in theCARs of the invention comprises (a) a HCVR having an amino acid sequenceselected from the group consisting of HCVR sequences listed in Table 1;and (b) a LCVR having an amino acid sequence selected from the groupconsisting of LCVR sequences listed in Table 1.

In one embodiments, the antigen-binding protein suitable for use in theCARs of the invention comprises (a) a HCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 20, 36,52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 236, 252, 268,284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492,508, and 524; (b) a HCDR2 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102,118, 134, 150, 166, 182, 198, 214, 222, 238, 254, 270, 286, 302, 318,334, 350, 366, 382, 398, 414, 430, 446, 462, 478, 494, 510, and 526; (c)a HCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152,168, 184, 200, 216, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368,384, 400, 416, 432, 448, 464, 480, 496, 512, and 528; (d) a LCDR1 domainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 228,244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452,468, 484, 500, 516, and 532; (e) a LCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 30, 46,62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262, 278, 294,310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518,and 534; and (f) a LCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 16. 32, 48, 64, 80, 96, 112,128, 144, 160, 176, 192, 208, 232, 248, 264, 280, 296, 312, 328, 344,360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, and 536.

In further embodiment, the antigen-binding protein suitable for use inthe CARs of the invention comprises a HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290,298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418,426/434, 442/450, 458/466, 474/482, 490/498, 506/514, and 522/530, suchas an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/10, 34/42, 82/90, 194/202, 282/290, and506/514.

In some embodiments the isolated antigen-binding protein for use in theCARs of the present invention is an scFv.

In other aspects, the present invention provides vectors comprising theisolated CAR nucleic acid molecules; and immune effector cellscomprising such vectors.

In yet other aspects of the present invention, methods for treating asubject having a HPV-associated disease or disorder, such as anHPV16E7-positive cancer, e.g., squamous cell carcinoma, e.g., cervicalcancer, head and neck small cell carcinoma, anogenital cancer, andoropharyngeal cancer are provided. The methods include administering tothe subject a population of immune effector cells comprising a CAR ofthe invention.

In some aspects, the present invention provides methods for detectingHPV16E7-positive cells, e.g., in a subject or in a sample obtained froma subject. The methods include contacting a cell, such as a cell sampleobtained from a subject, or administering to a subject, anantigen-binding protein of the invention comprising a detectable moiety,and detecting the presence of the detectable moiety.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The term “human papilloma virus” (“HPV”) refers to a small,non-enveloped deoxyribonucleic acid (DNA) virus that infects skin ormucosal cells. The circular, double-stranded viral genome isapproximately 8-kb in length. The genome encodes for 6 early proteinsresponsible for virus replication and 2 late proteins, L1 and L2, whichare the viral structural proteins. There are over 170 types of HPV thathave been identified, and they are designated by numbers. Some HPVtypes, such as HPV-5, may establish infections that persist for thelifetime of the individual without ever manifesting any clinicalsymptoms. HPV types 1 and 2 can cause common warts in some infectedindividuals. HPV types 6 and 11 can cause genital warts and respiratorypapillomatosis. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,59, 68, 73, and 82 are considered carcinogenic.

The term “HPV16E7” refers to the HPV type 16 early gene, designated E7,and the protein translated from the gene.

The amino acid sequence of full-length HPV16E7 is provided in GenBank asaccession number NP_041326.1 (SEQ ID NO: 537). The term “HPV16E7”includes recombinant HPV16E7 or a fragment thereof. The term alsoencompasses HPV16E7 or a fragment thereof coupled to, for example,histidine tag, mouse or human Fc, or a signal sequence such as ROR1. Incertain embodiments, the term comprises HPV16E7 or a fragment thereof inthe context of HLA-A2, linked to HLA-A2 or as displayed by HLA-A2.

The term “HLA” refers to the human leukocyte antigen (HLA) system orcomplex, which is a gene complex encoding the major histocompatibilitycomplex (MHC) proteins in humans. These cell-surface proteins areresponsible for the regulation of the immune system in humans. HLAscorresponding to MHC class I (A, B, and C) present peptides from insidethe cell.

The term “HLA-A” refers to the group of human leukocyte antigens (HLA)that are coded for by the HLA-A locus. HLA-A is one of three major typesof human MHC class I cell surface receptors. The receptor is aheterodimer, and is composed of a heavy a chain and smaller β chain. Theα chain is encoded by a variant HLA-A gene, and the β chain(β2-microglobulin) is an invariant β2 microglobulin molecule.

The term “HLA-A2” is one particular class I major histocompatibilitycomplex (MHC) allele group at the HLA-A locus; the α chain is encoded bythe HLA-A*02 gene and the β chain is encoded by the β2-microglobulin orB2M locus.

The term “antigen-binding protein,” “binding protein” or “bindingmolecule,” as used herein includes molecules that contain at least oneantigen-binding site that specifically binds to a molecule of interest,such as a conformational epitope of an HLA-A2 presented humanpapillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), e.g., aHLA-A2-displayed peptide comprising amino acid residues 11-19 or 82-90.A binding protein may be an antibody, such as a full-length antibody, oran antigen-binding fragment of an antibody, or a chimeric antigenreceptor (CAR), or any other polypeptide, e.g., a receptor-antibody(Rab) protein.

The term “HLA-A2:HPV16E7 antigen-binding protein” or “HLA-A2:HPV16E7antigen-binding protein,” or the like, refers to the an antigen-bindingprotein, such as an antibody, or antigen-binding portion thereof, thatspecifically binds to a conformational epitope by the presentation of apeptide fragment of HPV16E7, e.g., amino acid residues 11-19 or aminoacid residues 82-90), by HLA-A2. In certain embodiments, theconformational epitope is created on the surface of a cell by theHLA-A2-presented HPV16E7 peptide.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen-binding site in the variable region of anantigen-binding protein known as a paratope. A single antigen may havemore than one epitope. Thus, different antigen-binding proteins may bindto different areas on an antigen and may have different biologicaleffects. The term “epitope” also refers to a site on an antigen to whichB and/or T cells respond. It also refers to a region of an antigen thatis bound by an antigen-binding protein. Epitopes may be defined asstructural or functional. Functional epitopes are generally a subset ofthe structural epitopes and have those residues that directly contributeto the affinity of the interaction. Epitopes may also be“conformational,” that is, composed of non-linear amino acids. Incertain embodiments, epitopes may include determinants that arechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics.

In some embodiments of the invention, a binding protein is an antibody,or an antigen-binding fragment thereof, such as a full-length antibody,or antigen-binding fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen-binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antigen binding proteins, such as antibodies, havebeen described in the scientific literature in which one or two CDRs canbe dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139)analyzed the contact regions between antibodies and their antigens,based on published crystal structures, and concluded that only about onefifth to one third of CDR residues actually contact the antigen. Padlanalso found many antibodies in which one or two CDRs had no amino acidsin contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The anti-HLA-A2:HPV16E7 antigen-binding proteins, e.g., fully humananti-HLA-A2:HPV16E7 monoclonal antibodies, or antigen-binding fragmentsthereof, or CARs, disclosed herein may comprise one or more amino acidsubstitutions, insertions and/or deletions in the framework and/or CDRregions of the heavy and light chain variable domains as compared to thecorresponding germline sequences. Such mutations can be readilyascertained by comparing the amino acid sequences disclosed herein togermline sequences available from, for example, public antibody sequencedatabases. The present invention includes antigen-binding proteins,e.g., antibodies, or antigen-binding fragments thereof, or CARs, whichare derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antigen-binding protein was derived, or to thecorresponding residue(s) of another human germline sequence, or to aconservative amino acid substitution of the corresponding germlineresidue(s) (such sequence changes are referred to herein collectively as“germline mutations”). A person of ordinary skill in the art, startingwith the heavy and light chain variable region sequences disclosedherein, can easily produce numerous antigen-binding proteins, e.g.,antibodies, or antigen-binding fragments thereof, or CARs, whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which theantigen-binding protein, e.g., antibody, was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantigen-binding proteins, e.g., antibodies, or antigen-binding fragmentsthereof, or CARs, of the present invention may contain any combinationof two or more germline mutations within the framework and/or CDRregions, e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antigen-binding proteins, e.g.,antibodies and antigen-binding fragments, that contain one or moregermline mutations can be easily tested for one or more desired propertysuch as, improved binding specificity, increased binding affinity,improved or enhanced antagonistic or agonistic biological properties (asthe case may be), reduced immunogenicity, etc. Antigen binding proteins,e.g., antibodies, or antigen-binding fragments thereof, or CARs,obtained in this general manner are encompassed within the presentinvention.

The present invention also includes antigen-binding proteins, e.g.,fully human anti-HLA-A2:HPV16E7 monoclonal antibodies, orantigen-binding fragments thereof, or CARs, comprising variants of anyof the HCVR, LCVR, and/or CDR amino acid sequences disclosed hereinhaving one or more conservative substitutions. For example, the presentinvention includes anti-HLA-A2:HPV16E7 antigen-binding proteins havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human monoclonal antibodies(mAbs) of the invention may include amino acid residues not encoded byhuman germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo), for example in the CDRs and in particular CDR3. However, the term“human antibody”, as used herein, is not intended to include mAbs inwhich CDR sequences derived from the germline of another mammalianspecies (e.g., mouse), have been grafted onto human FR sequences. Theterm includes antibodies recombinantly produced in a non-human mammal,or in cells of a non-human mammal. The term is not intended to includeantibodies isolated from or generated in a human subject.

The term “recombinant”, as used herein, refers to antigen-bindingproteins, e.g., antibodies or antigen-binding fragments thereof, of theinvention created, expressed, isolated or obtained by technologies ormethods known in the art as recombinant DNA technology which include,e.g., DNA splicing and transgenic expression. The term refers toantigen-binding proteins, e.g., antibodies expressed in a non-humanmammal (including transgenic non-human mammals, e.g., transgenic mice),or a cell (e.g., CHO cells) expression system or isolated from arecombinant combinatorial human antibody library.

As used herein, the terms “chimeric antigen receptor” or “CAR”, usedinterchangeably herein, refer to a recombinant fused protein comprisingan extracellular domain capable of binding to an antigen (e.g., aconformational epitope of an HLA-A2 displayed HPV16E7 peptide, e.g., apeptide comprising amino acid residues 11-19 or 82-90 of HPV16E7), atransmembrane domain, and at least one intracellular signaling domain.

An “immune effector cell,” as used herein, refers to any cell of theimmune system that has one or more effector functions (e.g., cytotoxiccell killing activity, secretion of cytokines, induction of ADCC and/orCDC). In one embodiment, the immune effector cells used with the CARs asdescribed herein are T lymphocytes, in particular cytotoxic T cells(CTLs; CD8+ T cells) and helper T cells (HTLs; CD4+ T cells). Otherpopulations of T cells are also useful herein, for example naïve T cellsand memory T cells. As would be understood by the skilled person, othercells may also be used as immune effector cells with the CARs asdescribed herein. In particular, immune effector cells also include NKcells, NKT cells, neutrophils, and macrophages. Immune effector cellsalso include progenitors of effector cells wherein such progenitor cellscan be induced to differentiate into an immune effector cells in vivo orin vitro. Thus, in this regard, immune effector cell includesprogenitors of immune effectors cells such as hematopoietic stem cells(HSCs) contained within the CD34+ population of cells derived from cordblood, bone marrow or mobilized peripheral blood which uponadministration in a subject differentiate into mature immune effectorcells, or which can be induced in vitro to differentiate into matureimmune effector cells.

As disclosed herein, the term “off-target peptide” refers to a peptidethat differs by 1, 2, 3, 4, 5 or more amino acids from a target peptide(e.g., HPV16 E7 11-19 peptide). In certain embodiments, the termincludes a peptide that differs by less than or equal to 3 amino acidsthan the target peptide. For example, for a 9-mer peptide, if 1, 2, or 3amino acids are not identical to the target peptide, it is considered an“off-target” peptide. In certain embodiments, amino acid identity isexpressed in terms of ‘degree of similarity’ (DoS). If 6 or more aminoacids within a 9-mer peptide are identical, the DoS is 6. In certainembodiments, a peptide with DoS≤6 is considered an “off-target” peptide.The term “off-target” peptide also refers to a peptide that is similarto the target peptide based on sequence homology, is predicted to bindto HLA-A2 and is comprised in a protein that is expressed in essential,normal tissues.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antigen-binding protein, e.g., antibody, orantigen-binding fragments thereof, or CAR, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁸ M or less (e.g., a smaller K_(D) denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. As described herein,antigen-binding proteins, e.g., antibodies, have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically to aconformational epitope of an HLA-A2 presented human papillomavirus (HPV)16 E7 peptide (HPV16E7 peptide), e.g., a peptide comprising amino acidresidues 11-19 or 82-90 of HPV16E7.

The term “high affinity” antigen-binding protein, e.g., antibody, refersto those antigen-binding proteins, e.g., mAbs, having a binding affinityto conformational epitope of an HLA-A2 presented HPV16E7 peptide, e.g.,a peptide comprising amino acid residues 11-19 or 82-90 of HPV16E7,expressed as K_(D), of at least 10⁻⁸ M; preferably 10⁻⁹M; morepreferably 10⁻¹⁰M, even more preferably 10⁻¹¹ M, even more preferably10⁻¹² M, as measured by surface plasmon resonance, e.g., BIACORE™ orsolution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antigen-bindingprotein that dissociates from HLA-A2:HPV16E7, with a rate constant of1×10⁻³ s⁻¹ or less, preferably 1×10⁻⁴ s⁻¹ or less, as determined bysurface plasmon resonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antigen-binding protein (e.g.,antibody), “antigen-binding fragment” of an antigen-binding protein(e.g., antibody), and the like, as used herein, include any naturallyoccurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind toconformational epitope of an HLA-A2 presented HPV16E7 peptide, e.g., apeptide comprising amino acid residues 11-19 or 82-90 of HPV16E7 coupledto HLA-A2.

In specific embodiments, antigen-binding proteins, e.g., antibody orantibody fragments, or CARs, of the invention may be conjugated to amoiety such as a ligand, a detectable moiety, or a therapeutic moiety(“immunoconjugate”), such as a cytotoxin, a second anti-HLA-A2:HPV16E7antigen-binding protein, an antibody to a tumor-specific antigen, ananti-cancer drug, or any other therapeutic moiety useful for treating adisease or condition including HPV-associated disease or disorder, suchas an HPV16E7-positive cancer or HPV infection including chronic HPVinfection.

An “isolated antigen-binding protein”, e.g., an isolated antibody, asused herein, is intended to refer to an antigen-binding protein, e.g.,antibody, that is substantially free of other antigen-binding proteins,e.g., antibodies (Abs), having different antigenic specificities (e.g.,an isolated antibody that specifically binds HLA-A2:HPV16E7, or afragment thereof, is substantially free of antigen-binding proteins,e.g., antibodies, that specifically bind antigens other than aconformational epitope of an HLA-A2 presented HPV16E7 peptide.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antigen-bindingprotein-antigen interaction.

The term “cross-competes”, as used herein, means an antigen-bindingprotein, e.g., antibody or antigen-binding fragment thereof, binds to anantigen and inhibits or blocks the binding of another antigen-bindingprotein, e.g., antibody or antigen-binding fragment thereof. The termalso includes competition between two antigen-binding proteins, e.g.,antibodies, in both orientations, i.e., a first antigen-binding protein,e.g., antibody, that binds and blocks binding of second antigen-bindingprotein, e.g., antibody, and vice-versa. In certain embodiments, thefirst antigen-binding protein, e.g., antibody, and secondantigen-binding protein, e.g., antibody, may bind to the same epitope.Alternatively, the first and second antigen-binding proteins, e.g.,antibodies, may bind to different, but overlapping epitopes such thatbinding of one inhibits or blocks the binding of the second, e.g., viasteric hindrance. Cross-competition between antigen-binding proteins,e.g., antibodies, may be measured by methods known in the art, forexample, by a real-time, label-free bio-layer interferometry assay.Cross-competition between two antigen-binding proteins, e.g.,antibodies, may be expressed as the binding of the secondantigen-binding protein, e.g., antibody, that is less than thebackground signal due to self-self binding (wherein first and secondantigen-binding proteins, e.g., antibodies, is the same antigen-bindingprotein, e.g., antibody). Cross-competition between 2 antigen-bindingproteins, e.g., antibodies, may be expressed, for example, as % bindingof the second antigen-binding protein, e.g., antibody, that is less thanthe baseline self-self background binding (wherein first and secondantigen-binding proteins, e.g., antibodies is the same antigen-bindingprotein, e.g., antibody).

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, as discussedbelow. A nucleic acid molecule having substantial identity to areference nucleic acid molecule may, in certain instances, encode apolypeptide having the same or substantially similar amino acid sequenceas the polypeptide encoded by the reference nucleic acid molecule.

Sequence identity can be calculated using an algorithm, for example, theNeedleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48:443-453) for global alignment, or the Smith Waterman algorithm (Smithand Waterman 1981, J. Mol. Biol. 147: 195-197) for local alignment.Another preferred algorithm is described by Dufresne et al in NatureBiotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the softwareGenePAST (GQ Life Sciences, Inc. Boston, Mass.).

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 96%, 97%, 98% or 99% sequence identity. Preferably, residuepositions, which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, the term “subject” refers to an animal, preferably amammal, in need of amelioration, prevention and/or treatment of adisease or disorder such as HPV infection, or a HPV-associated diseaseor disorder, such as a HPV-associated cancer (e.g., an HPV16E7-positivecancer). The term includes human subjects who have or are at risk ofhaving HPV-associated disease or disorder, such as an HPV-associatedcancer, metastatic HPV-associated cancer or HPV infection.

As used herein, “anti-cancer drug” means any agent useful to treat orameliorate or inhibit cancer including, but not limited to, cytotoxinsand agents such as antimetabolites, alkylating agents, anthracyclines,antibiotics, antimitotic agents, procarbazine, hydroxyurea,asparaginase, corticosteroids, cyclophosphamide, mytotane (O,P′-(DDD)),biologics (e.g., antibodies and interferons) and radioactive agents. Asused herein, “a cytotoxin or cytotoxic agent”, also refers to achemotherapeutic agent and means any agent that is detrimental to cells.Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B,gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin,etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

As used herein, the term “anti-viral drug” refers to any drug or therapyused to treat, prevent, or ameliorate a viral infection in a hostsubject. The term “anti-viral drug” includes, but is not limited tozidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz,cobicistat, tenofovir, rilpivirine, analgesics and corticosteroids.

An immunogen comprising any one of the following can be used to generateantigen-binding proteins, e.g., antibodies, to a conformational epitopeof an HLA-A2 presented HPV16E7 peptide, e.g., a peptide comprising aminoacid residues 11-19 or residues 82-90 of HPV16E7 linked to HLA-A2. Incertain embodiments, the antigen-binding proteins, e.g., antibodies, ofthe invention are obtained from mice immunized with a full length nativeHPV16E7 protein (See NCBI accession number NP_041326.1) (SEQ ID NO: 537)or with a recombinant HPV16E7 peptide, such as a peptide comprisingeither amino acids residues 11-19 (YMLDLQPET; SEQ ID NO: 538) of GenBankAccession NP_041326.1 (SEQ ID NO: 537) or amino acid residues 82-90(LLMGTLGIV; SEQ ID NO: 539) of GenBank Accession NP_041326.1 (SEQ ID NO:537), linked to HLA-A2.

Alternatively, HPV16E7 or a fragment thereof may be produced usingstandard biochemical techniques and modified in the context of HLA-A2and used as immunogen.

In some embodiments, the immunogen may be a recombinant HPV16E7 peptideexpressed in E. coli or in any other eukaryotic or mammalian cells suchas Chinese hamster ovary (CHO) cells.

In certain embodiments, antigen-binding proteins that bind specificallya conformational epitope of an HLA-A2 presented HPV16E7 peptide may beprepared using fragments of the above-noted regions, or peptides thatextend beyond the designated regions by about 5 to about 20 amino acidresidues from either, or both, the N or C terminal ends of the regionsdescribed herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationof HLA-A2:HPV16E7 specific antigen-binding proteins, e.g., antibodies.

The peptides may be modified to include addition or substitution ofcertain residues for tagging or for purposes of conjugation to carriermolecules, such as, KLH. For example, a cysteine may be added at eitherthe N terminal or C terminal end of a peptide, or a linker sequence maybe added to prepare the peptide for conjugation to, for example, KLH forimmunization.

Non-limiting, exemplary in vitro assays for measuring binding activityare illustrated in Examples herein. In Example 4, the binding affinitiesand kinetic constants of human anti-HLA-A2:HPV16E7 specificantigen-binding proteins, e.g., antibodies were determined by surfaceplasmon resonance and the measurements were conducted on a Biacore 4000or T200 instrument. Examples 6 and 7 describe the binding of theantibodies to cells overexpressing fragments of HPV16E7.

The antigen-binding proteins, e.g., antibodies, specific forHLA-A2:HPV16E7 may contain no additional labels or moieties, or they maycontain an N-terminal or C-terminal label or moiety. In one embodiment,the label or moiety is biotin. In a binding assay, the location of alabel (if any) may determine the orientation of the peptide relative tothe surface upon which the peptide is bound. For example, if a surfaceis coated with avidin, a peptide containing an N-terminal biotin will beoriented such that the C-terminal portion of the peptide will be distalto the surface. In one embodiment, the label may be a radionuclide, afluorescent dye or a MRI-detectable label. In certain embodiments, suchlabeled antigen-binding proteins may be used in diagnostic assaysincluding imaging assays.

Antigen Binding Proteins

The present invention provides antigen-binding proteins that includeantibodies, or antigen-binding fragments thereof, and CARs (e.g.,nucleic acid molecules encoding a CAR of the invention) (describedbelow). Unless specifically indicated otherwise, the term “antibody,” asused herein, shall be understood to encompass antibody moleculescomprising two immunoglobulin heavy chains and two immunoglobulin lightchains (i.e., “full antibody molecules”) as well as antigen-bindingfragments thereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding fragment”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a conformational epitope of an HLA-A2 presented HPV16E7 peptide.An antigen-binding protein, such as an antibody fragment, may include aFab fragment, a F(ab′)₂ fragment, a Fv fragment, a dAb fragment, afragment containing a CDR, or an isolated CDR. Antigen binding proteins,such as antigen-binding fragments of an antibody, may be derived, e.g.,from full antibody molecules using any suitable standard techniques suchas proteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and (optionally) constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments of an antibody,include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments;(iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAbfragments; and (vii) minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region of an antibody (e.g.,an isolated complementarity determining region (CDR) such as a CDR3peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineeredmolecules, such as domain-specific antibodies, single domain antibodies,domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies,diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g.monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antigen-binding protein (e.g.,antibody), will typically comprise at least one variable domain. Thevariable domain may be of any size or amino acid composition and willgenerally comprise at least one CDR, which is adjacent to or in framewith one or more framework sequences. In antigen-binding proteins havinga V_(H) domain associated with a V_(L) domain, the V_(H) and V_(L)domains may be situated relative to one another in any suitablearrangement. For example, the variable region may be dimeric and containV_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, theantigen-binding fragment of an antibody, may contain a monomeric V_(H)or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody, maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antigen-binding protein of the present invention include: (i)V_(H)-C_(H)1; (ii) V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv)V_(H)-C_(H)1-C_(H)2; (v) V_(H)-C_(H)1-C_(H)2-C_(H)3;V_(H)-C_(H)2-C_(H)3; V_(H)-C_(L); V_(L)-C_(H)1; (ix) V_(L)- C_(H)2; (X)V_(L)-C_(H)3; (xi) V_(L)-C_(H)1-C_(H)2; (XII)V_(L)-C_(H)1-C_(H)2-C_(H)3; V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L).In any configuration of variable and constant domains, including any ofthe exemplary configurations listed above, the variable and constantdomains may be either directly linked to one another or may be linked bya full or partial hinge or linker region. A hinge region may consist ofat least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, whichresult in a flexible or semi-flexible linkage between adjacent variableand/or constant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody, of the present invention maycomprise a homo-dimer or heterodimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding proteins, e.g.,antigen-binding fragments of an antibody, may be mono-specific ormulti-specific (e.g., bi-specific). A multi-specific antigen-bindingfragment of an antibody will typically comprise at least two differentvariable domains, wherein each variable domain is capable ofspecifically binding to a separate antigen or to a different epitope onthe same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Antigen-Binding Proteins

Methods for generating antigen-binding proteins, such as humanantibodies, in transgenic mice are known in the art. Any such knownmethods can be used in the context of the present invention to makehuman antibodies that specifically bind to a conformational epitope ofan HLA-A2 presented human papillomavirus (HPV) 16 E7 peptide (HPV16E7peptide).

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating antigen-binding proteins, e.g., monoclonalantibodies, high affinity antigen-binding proteins, e.g., chimericantibodies, to conformational epitope of an HLA-A2 presented HPV16E7peptide, are initially isolated having a human variable region and amouse constant region. The VELOCIMMUNE® technology involves generationof a transgenic mouse having a genome comprising human heavy and lightchain variable regions operably linked to endogenous mouse constantregion loci such that the mouse produces an antigen-binding protein,e.g., antibody, comprising a human variable region and a mouse constantregion in response to antigenic stimulation. The DNA encoding thevariable regions of the heavy and light chains of the antibody areisolated and operably linked to DNA encoding the human heavy and lightchain constant regions. The DNA is then expressed in a cell capable ofexpressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antigen-binding proteins, e.g., antibodies. Thelymphatic cells may be fused with a myeloma cell line to prepareimmortal hybridoma cell lines, and such hybridoma cell lines arescreened and selected to identify hybridoma cell lines that produceantibodies specific to the antigen of interest. DNA encoding thevariable regions of the heavy chain and light chain may be isolated andlinked to desirable isotypic constant regions of the heavy chain andlight chain. Such an antigen-binding protein may be produced in a cell,such as a CHO cell. Alternatively, DNA encoding the antigen-specificantigen-binding proteins, e.g., chimeric antibodies, or the variabledomains of the light and heavy chains may be isolated directly fromantigen-specific lymphocytes.

Initially, high affinity antigen-binding proteins, e.g., chimericantibodies, are isolated having a human variable region and a mouseconstant region. As in the experimental section below, theantigen-binding proteins are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Themouse constant regions are replaced with a desired human constant regionto generate the antigen-binding proteins, e.g., fully human antibodies,of the invention, for example wild-type or modified IgG1 or IgG4. Whilethe constant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

Bioequivalents

The anti-HLA-A2:HPV16E7 antigen-binding proteins of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antigen-binding proteins, e.g., antibodies, butthat retain the ability to bind a conformational epitope of an HLA-A2presented HPV16E7 peptide. Such variant antigen-binding proteinscomprise one or more additions, deletions, or substitutions of aminoacids when compared to parent sequence, but exhibit biological activitythat is essentially equivalent to that of the described antigen-bindingproteins. Likewise, the antigen-binding protein-encoding DNA sequencesof the present invention encompass sequences that comprise one or moreadditions, deletions, or substitutions of nucleotides when compared tothe disclosed sequence, but that encode an antigen-binding protein thatis essentially bioequivalent to an antigen-binding protein of theinvention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single dose or multipledoses. Some antigen-binding proteins or antibodies will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins (or antibodies) arebioequivalent if there are no clinically meaningful differences in theirsafety, purity, or potency.

In one embodiment, two antigen-binding proteins (or antibodies) arebioequivalent if a patient can be switched one or more times between thereference product and the biological product without an expectedincrease in the risk of adverse effects, including a clinicallysignificant change in immunogenicity, or diminished effectiveness, ascompared to continued therapy without such switching.

In one embodiment, two antigen-binding proteins (or antibodies) arebioequivalent if they both act by a common mechanism or mechanisms ofaction for the condition or conditions of use, to the extent that suchmechanisms are known.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antigen-binding proteinor its metabolites is measured in blood, plasma, serum, or otherbiological fluid as a function of time; (b) an in vitro test that hasbeen correlated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of theantigen-binding protein (or its target) is measured as a function oftime; and (d) in a well-controlled clinical trial that establishessafety, efficacy, or bioavailability or bioequivalence of anantigen-binding protein.

Bioequivalent variants of the antigen-binding proteins (or antibodies)of the invention may be constructed by, for example, making varioussubstitutions of residues or sequences or deleting terminal or internalresidues or sequences not needed for biological activity. For example,cysteine residues not essential for biological activity can be deletedor replaced with other amino acids to prevent formation of unnecessaryor incorrect intramolecular disulfide bridges upon renaturation. Inother contexts, bioequivalent antigen-binding proteins may includeantigen-binding protein variants comprising amino acid changes, whichmodify the glycosylation characteristics of the antigen-bindingproteins, e.g., mutations that eliminate or remove glycosylation.

Anti-HLA-A2:HPV16E7 Antigen Binding-Proteins Comprising Fc Variants

According to certain embodiments of the present invention,anti-HLA-A2:HPV16E7 antigen-binding proteins, e.g., antibodies, areprovided comprising an Fc domain comprising one or more mutations whichenhance or diminish antigen-binding protein binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-HLA-A2:HPV16E7 antigen-binding proteinscomprising a mutation in the C_(H)2 or a C_(H)3 region of the Fc domain,wherein the mutation(s) increases the affinity of the Fc domain to FcRnin an acidic environment (e.g., in an endosome where pH ranges fromabout 5.5 to about 6.0). Such mutations may result in an increase inserum half-life of the antigen-binding protein when administered to ananimal. Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Qor K) and/or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F orN434Y]); or a modification at position 250 and/or 428; or a modificationat position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment,the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P). In yet another embodiment, themodification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A)modification.

For example, the present invention includes anti-HLA-A2:HPV16E7antigen-binding proteins comprising an Fc domain comprising one or morepairs or groups of mutations selected from the group consisting of: 250Qand 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y,S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I(e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380Aand N434A); and 433K and 434F (e.g., H433K and N434F). In oneembodiment, the present invention includes anti-HLA-A2:HPV16E7antigen-binding proteins comprising an Fc domain comprising a S108Pmutation in the hinge region of IgG4 to promote dimer stabilization. Allpossible combinations of the foregoing Fc domain mutations, and othermutations within the antigen-binding protein variable domains disclosedherein, are contemplated within the scope of the present invention.

The present invention also includes anti-HLA-A2:HPV16E7 antigen-bindingproteins comprising a chimeric heavy chain constant (C_(H)) region,wherein the chimeric C_(H) region comprises segments derived from theC_(H) regions of more than one immunoglobulin isotype. For example, theantigen-binding proteins of the invention may comprise a chimeric C_(H)region comprising part or all of a C_(H)2 domain derived from a humanIgG1, human IgG2 or human IgG4 molecule, combined with part or all of aC_(H)3 domain derived from a human IgG1, human IgG2 or human IgG4molecule. According to certain embodiments, the antigen-binding proteinsof the invention comprise a chimeric C_(H) region having a chimerichinge region. For example, a chimeric hinge may comprise an “upperhinge” amino acid sequence (amino acid residues from positions 216 to227 according to EU numbering) derived from a human IgG1, a human IgG2or a human IgG4 hinge region, combined with a “lower hinge” sequence(amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antigen-binding protein comprising a chimeric C_(H) region asdescribed herein may, in certain embodiments, exhibit modified Fceffector functions without adversely affecting the therapeutic orpharmacokinetic properties of the antigen-binding protein. (See, e.g.,U.S. Patent Publication No. 20140243504, the disclosure of which ishereby incorporated by reference in its entirety).

Biological Characteristics of the Antigen-Binding Proteins

In general, the antigen-binding proteins of the present inventionfunction by binding to a conformational epitope of an HLA-A2 presentedhuman papillomavirus (HPV) 16 E7 peptide (HPV16E7) peptide.

The present invention includes anti-HLA-A2:HPV16E7 antigen-bindingproteins that bind HPV16E7 peptide in the context of HLA-A2 with highspecificity. The anti-HLA-A2:HPV16E7 antigen-binding proteins do notbind to the HPV16E7 peptide in the absence of HLA-A2. Further, theanti-HLA-A2:HPV16E7 antigen-binding proteins do not bind to anoff-target peptide in the context of HLA-A2.

The present invention includes anti-HLA-A2:HPV16E7 antigen-bindingproteins that bind monomeric HLA-A2:HPV16E7 11-19 peptide with highaffinity. For example, the present invention includes antigen-bindingproteins that bind monomeric HLA-A2:HPV16E7 11-19 peptide (e.g., at 25°C. or at 37° C.) with a K_(D) of less than about 20 nM as measured bysurface plasmon resonance, e.g., using the assay format as defined inExample 4 herein. In certain embodiments, the antigen-binding proteinsbind monomeric HLA-A2:HPV16E7 11-19 peptide with a K_(D) of less thanabout 15 nM, less than about 12 nM, less than about 10 nM, less thanabout 5 nM, less than about 2 nM, less than about 1 nM, less than about0.5 nM less than about 0.1 nM, less than about 0.05 nM or less thanabout 0.04 nM, as measured by surface plasmon resonance, e.g., using theassay format as defined in Example 4 herein, or a substantially similarassay.

The present invention includes antigen-binding proteins that bindmonomeric HLA-A2:HPV16E7 82-90 peptide (e.g., at 25° C. or at 37° C.)with a K_(D) of less than about 25 nM as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 4 herein,or a substantially similar assay. In certain embodiments, theantigen-binding proteins bind monomeric HLA-A2:HPV16E7 82-90 peptidewith a K_(D) of less than about 20 nM, less than about 15 nM, less thanabout 12 nM, less than about 10 nM, less than about 5 nM, less thanabout 2 nM, less than about 1 nM, less than about 0.5 nM less than about0.1 nM, less than about 0.05 nM or less than about 0.04 nM, as measuredby surface plasmon resonance, e.g., using the assay format as defined inExample 4 herein, or a substantially similar assay.

The present invention also includes antigen-binding proteins that bindto a cell expressing an HLA-A2:HPV16E7 11-19 peptide with an EC₅₀ lessthan about 6 nM and do not bind to cells expressing predicted off-targetpeptides as determined by luminescence assay, as defined in Example 6herein, or a substantially similar assay. In certain embodiments, theantigen-binding proteins bind to a cell expressing an HLA-A2:HPV16E711-19 peptide with an EC₅₀ less than about less than about 6 nM, lessthan about 5 nM, less than about 2 nM, less than about 1 nM, or lessthan about 0.5 nM, and do not bind to cells expressing predictedoff-target peptides as determined by luminescence assay, as defined inExample 6 herein, or a substantially similar assay. e.g., using theassay format in Example 6 herein, or a substantially similar assay.

The present invention also includes antigen-binding proteins that bindto a cell expressing an HLA-A2:HPV16E7 82-90 peptide with an EC₅₀ lessthan about 1 nM and do not bind to cells expressing predicted off-targetpeptides as determined by luminescence assay, as defined in Example 6herein, or a substantially similar assay. In certain embodiments, theantigen-binding proteins bind to a cell expressing an HLA-A2:HPV16E782-90 peptide with an EC₅₀ less than about less than about 1 nM, lessthan about 0.5 nM, less than about 0.2 nM, or less than about 0.01 nMand do not bind to cells expressing predicted off-target peptides asdetermined by luminescence assay, as defined in Example 6 herein, or asubstantially similar assay. e.g., using the assay format in Example 6herein, or a substantially similar assay.

The present invention also includes antigen-binding proteins that bindto a cell expressing an HLA-A2:HPV16E7 11-19 peptide with an EC₅₀ lessthan about 30 nM as measured by a flow cytometry assay as defined inExample 7 herein, or a substantially similar assay. In certainembodiments, the antigen-binding proteins bind to a cell expressing anHLA-A2:HPV16E7 11-19 peptide with an EC₅₀ less than about 25 nM, lessthan about 20 nM, less than about 15 nM, less than about 10 nM, lessthan about 5 nM, less than about 2 nM, less than about 1 nM, or lessthan about 0.5 nM, as measured by a flow cytometry assay, e.g., usingthe assay format in Example 7 herein, or a substantially similar assay.

The present invention also includes antigen-binding proteins that bindto a cell expressing an HLA-A2:HPV16E7 82-90 peptide with an EC₅₀ lessthan about 75 nM as measured by a flow cytometry assay as defined inExample 7 herein, or a substantially similar assay. In certainembodiments, the antigen-binding proteins bind to a cell expressing anHLA-A2:HPV16E7 82-90 peptide with an EC₅₀ less than about 75 nM, lessthan about 70 nM, less than about 65 nM, less than about 60 nM, lessthan about 55 nM, less than about 50 nM, less than about 45 nM, lessthan about 40 nM, less than about 35 nM, less than about 30 nM, lessthan about 25 nM, less than about 20 nM, less than about 15 nM, lessthan about 10 nM, less than about 5 nM, less than about 2 nM, less thanabout 1 nM, or less than about 0.5 nM, as measured by a flow cytometryassay, e.g., using the assay format in Example 7 herein, or asubstantially similar assay.

In certain embodiments, the antigen-binding proteins of the presentinvention are useful in inhibiting the growth of a tumor or delaying theprogression of cancer when administered prophylactically to a subject inneed thereof and may increase survival of the subject. For example, theadministration of an antigen-binding protein of the present inventionmay lead to shrinking of a primary tumor and may prevent metastasis ordevelopment of secondary tumors. In certain embodiments, theantigen-binding proteins of the present invention are useful ininhibiting the growth of a tumor when administered therapeutically to asubject in need thereof and may increase survival of the subject. Forexample, the administration of a therapeutically effective amount of anantigen-binding protein of the invention to a subject may lead toshrinking and disappearance of an established tumor in the subject.

In one embodiment, the invention provides an isolated recombinantantigen-binding protein thereof that binds to a conformational epitopeof an HLA-A2 presented HPV16E7 peptide, wherein the antigen-bindingprotein exhibits one or more of the following characteristics: (i)comprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, 210, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378,394, 410, 426, 442, 458, 474, 490, 506, and 522, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% sequence identity; (ii)comprises a LCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,170, 186, 202, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386,402, 418, 434, 450, 466, 482, 498, 514, and 530, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (iii) comprises a HCDR3 domain havingan amino acid sequence selected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224, 240,256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464,480, 496, 512, and 528, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity; and a LCDR3 domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 16,32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248, 264,280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488,504, 520, and 536, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity; (iv) comprises a HCDR1 domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 4,20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 236,252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460,476, 492, 508, and 524, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity; a LCDR1 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 12, 28,44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 228, 244, 260, 276,292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500,516, and 532, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity; and a LCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 14, 30, 46,62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262, 278, 294,310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518,and 534, or a substantially similar sequence thereof having at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity; (v) binds monomeric HLA-A2:HPV16E7 11-19 peptidewith a binding dissociation equilibrium constant (KD) of less than about20 nM as measured in a surface plasmon resonance assay at 25° C.; (vi)binds monomeric HLA-A2:HPV16E7 82-90 peptide with a binding dissociationequilibrium constant (KD) of less than about 25 nM as measured in asurface plasmon resonance assay at 25° C.; (vii) binds to HLA-A2:HPV16E711-19 peptide expressing cells with an EC50 less than about 6 nM and donot bind to cells expressing predicted off-target peptides as determinedby luminescence assay; (viii) binds to HLA-A2:HPV16E7 82-90 peptideexpressing cells with an EC50 less than about 1 nM and do notsubstantially bind to cells expressing predicted off-target peptides asdetermined by luminescence assay; (ix) binds to HLA-A2:HPV16E7 11-19peptide expressing cells with an EC50 less than about 30 nM asdetermined by flow cytometry assay; (x) binds to HLA-A2:HPV16E7 82-90peptide expressing cells with an EC50 less than about 75 nM asdetermined by flow cytometry assay; (xi) does not bind to aHLA-A2-displayed off-target peptide wherein the peptide differs by 1, 2,3, 4, 5 or more amino acids from SEQ ID NO: 538; and (xii) does not bindto a HLA-A2-displayed off-target peptide wherein the peptide differs by1, 2, 3, 4, 5 or more amino acids from SEQ ID NO: 539.

The antigen-binding proteins of the present invention may possess one ormore of the aforementioned biological characteristics, or anycombinations thereof. Other biological characteristics of theantigen-binding proteins of the present invention will be evident to aperson of ordinary skill in the art from a review of the presentdisclosure including the working Examples herein.

Epitope Mapping and Related Technologies

The present invention includes anti-HLA-A2:HPV16E7 antigen-bindingproteins which interact with one or more amino acids found within one ormore domains of the HLA-A2 displayed HPV16E7 peptide. The epitope mayconsist of a plurality of non-contiguous amino acids (or amino acidsequences) located within either or both of the aforementioned domainsof the HPV16E7 molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding protein “interacts with oneor more amino acids” within a polypeptide or protein. Exemplarytechniques include, for example, routine cross-blocking assays, such asthat described in Antibodies, Harlow and Lane (Cold Spring Harbor Press,Cold Spring Harbor, N.Y.). Other methods include alanine scanningmutational analysis, peptide blot analysis (Reineke (2004) Methods Mol.Biol. 248: 443-63), peptide cleavage analysis crystallographic studiesand NMR analysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which anantigen-binding protein interacts is hydrogen/deuterium exchangedetected by mass spectrometry. In general terms, the hydrogen/deuteriumexchange method involves deuterium-labeling the protein of interest,followed by binding the antigen-binding protein to the deuterium-labeledprotein. Next, the protein/antigen-binding protein complex istransferred to water and exchangeable protons within amino acids thatare protected by the antigen-binding protein complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antigen-bindingprotein interface may retain deuterium and therefore exhibit relativelyhigher mass compared to amino acids not included in the interface. Afterdissociation of the antigen-binding protein, the target protein issubjected to protease cleavage and mass spectrometry analysis, therebyrevealing the deuterium-labeled residues which correspond to thespecific amino acids with which the antigen-binding protein interacts.See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen andSmith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antigen-binding proteins, e.g., antibodies(mAbs), directed against the same antigen according to the similaritiesof the binding profile of each antibody to chemically or enzymaticallymodified antigen surfaces (see US 2004/0101920, herein specificallyincorporated by reference in its entirety). Each category may reflect aunique epitope either distinctly different from or partially overlappingwith epitope represented by another category. This technology allowsrapid filtering of genetically identical antigen-binding proteins, suchthat characterization can be focused on genetically distinctantigen-binding proteins. When applied to hybridoma screening, MAP mayfacilitate identification of rare hybridoma clones that produceantigen-binding proteins having the desired characteristics. MAP may beused to sort the antigen-binding proteins of the invention into groupsof antigen-binding proteins binding different epitopes.

The present invention includes anti-HLA-A2:HPV16E7 antigen-bindingproteins that bind to the same epitope, or a portion of the epitope, asany of the specific exemplary antigen-binding proteins described hereinin Table 1, or an antigen-binding protein having the CDR sequences ofany of the exemplary antigen-binding proteins described in Table 1.Likewise, the present invention also includes anti-HLA-A2:HPV16E7antigen-binding proteins that compete for binding to HLA-A2:HPV16E7 or afragment thereof with any of the specific exemplary antigen-bindingproteins described herein in Table 1, or an antigen-binding proteinhaving the CDR sequences of any of the exemplary antigen-bindingproteins described in Table 1.

One can easily determine whether an antigen-binding protein binds to thesame epitope as, or competes for binding with, a referenceanti-HLA-A2:HPV16E7 antigen-binding protein by using routine methodsknown in the art. For example, to determine if a test antigen-bindingprotein binds to the same epitope as a reference anti-HLA-A2:HPV16E7antigen-binding protein of the invention, the reference antigen-bindingprotein is allowed to bind to a HLA-A2:HPV16E7 protein or peptide undersaturating conditions. Next, the ability of a test antigen-bindingprotein to bind to the HLA-A2:HPV16E7 molecule is assessed. If the testantigen-binding protein is able to bind to HLA-A2:HPV16E7 followingsaturation binding with the reference anti-HLA-A2:HPV16E7antigen-binding protein, it can be concluded that the testantigen-binding protein binds to a different epitope than the referenceanti-HLA-A2:HPV16E7 antigen-binding protein. On the other hand, if thetest antigen-binding protein is not able to bind to the HLA-A2:HPV16E7protein following saturation binding with the referenceanti-HLA-A2:HPV16E7 antigen-binding protein, then the testantigen-binding protein may bind to the same epitope as the epitopebound by the reference anti-HLA-A2:HPV16E7 antigen-binding protein ofthe invention.

To determine if an antigen-binding protein competes for binding with areference anti-HLA-A2:HPV16E7 antigen-binding protein, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding protein is allowed tobind to a HLA-A2:HPV16E7 protein under saturating conditions followed byassessment of binding of the test antigen-binding protein to theHLA-A2:HPV16E7 molecule. In a second orientation, the testantigen-binding protein is allowed to bind to a HLA-A2:HPV16E7 moleculeunder saturating conditions followed by assessment of binding of thereference antigen-binding protein to the HLA-A2:HPV16E7 molecule. If, inboth orientations, only the first (saturating) antigen-binding proteinis capable of binding to the HLA-A2:HPV16E7 molecule, then it isconcluded that the test antigen-binding protein and the referenceantigen-binding protein compete for binding to HLA-A2:HPV16E7. As willbe appreciated by a person of ordinary skill in the art, anantigen-binding protein that competes for binding with a referenceantigen-binding protein may not necessarily bind to the identicalepitope as the reference antigen-binding protein, but may stericallyblock binding of the reference antigen-binding protein by binding anoverlapping or adjacent epitope.

Two antigen-binding proteins bind to the same or overlapping epitope ifeach competitively inhibits (blocks) binding of the other to theantigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of oneantigen-binding protein inhibits binding of the other by at least 50%but preferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502).Alternatively, two antigen-binding proteins have the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antigen-binding protein reduce or eliminatebinding of the other. Two antigen-binding proteins have overlappingepitopes if some amino acid mutations that reduce or eliminate bindingof one antigen-binding protein reduce or eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antigen-binding protein is in fact due to bindingto the same epitope as the reference antigen-binding protein or ifsteric blocking (or another phenomenon) is responsible for the lack ofobserved binding. Experiments of this sort can be performed using ELISA,RIA, surface plasmon resonance, flow cytometry or any other quantitativeor qualitative antigen-binding protein-binding assay available in theart.

Immunoconjugates

The invention encompasses anti-HLA-A2:HPV16E7 antigen-binding proteinsconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin or a chemotherapeutic agent to treat cancer. As used herein,the term “immunoconjugate” refers to an antigen-binding protein which ischemically or biologically linked to a cytotoxin, a radioactive agent, acytokine, an interferon, a target or reporter moiety, such as adetectable moiety, an enzyme, a toxin, a peptide or protein or atherapeutic agent. The antigen-binding protein may be linked to thecytotoxin, radioactive agent, cytokine, interferon, target or reportermoiety, enzyme, toxin, peptide or therapeutic agent at any locationalong the molecule so long as it is able to bind its target. Examples ofimmunoconjugates include antigen-binding protein-drug conjugates andantigen-binding protein-toxin fusion proteins. In one embodiment, theagent may be a second different antibody to HPV16E7 or HLA-A2:HPV16E7.In certain embodiments, the antigen-binding protein may be conjugated toan agent specific for a tumor cell or a virally infected cell, i.e., anHPV infected cell. The type of therapeutic moiety that may be conjugatedto the anti-HLA-A2:HPV16E7 antigen-binding protein and will take intoaccount the condition to be treated and the desired therapeutic effectto be achieved. Examples of suitable agents for forming immunoconjugatesare known in the art; see for example, PCT Publication No. WO 05/103081.

Chimeric Antigen Receptors (CAR)

Chimeric antigen receptors (CARs) redirect T cell specificity towardantibody-recognized antigens expressed on the surface of cancer cells,while T cell receptors (TCRs) extend the range of targets to includeintracellular tumor antigens. CAR redirected T cells specific for the Bcell differentiation antigen CD19 have shown dramatic efficacy in thetreatment of B cell malignancies, while TCR-redirected T cells haveshown benefits in patients suffering from solid cancer. Stauss et al.describe strategies to modify therapeutic CARs and TCRs, for use in thetreatment of cancer, for example, to enhance the antigen-specificeffector function and limit toxicity of engineered T cells (CurrentOpinion in Pharmacology 2015, 24:113-118).

One aspect of the invention includes a chimeric antigen receptor (CAR)which is specific for an HPV16E7 peptide displayed on the surface oftumor cells by HLA-A2, such as a peptide comprising amino acid residues11-19 or 82-90 of HPV16E7. In one embodiment of the present invention, aCAR as described herein comprises an extracellular target-specificbinding domain, a transmembrane domain, an intracellular signalingdomain (such as a signaling domain derived from CD3zeta or FcRgamma),and/or one or more co-stimulatory signaling domains derived from aco-stimulatory molecule, such as, but not limited to, CD28, CD137, CD134or CD278. In one embodiment, the CAR includes a hinge or spacer regionbetween the extracellular binding domain and the transmembrane domain,such as a CD8alpha hinge. In another embodiment of the presentinvention, a CAR as described herein comprises an extracellulartarget-specific binding domain, and a T cell receptor constant domain(“T-body construct”).

It is to be understood that, for use in any of the CARs describedherein, the extracellular target-specific binding domain may comprise aFab, a Fab′, a (Fab′)2, an Fv, or a single chain Fv (scFv) of anantigen-binding protein of the invention.

As used herein, the binding domain or the extracellular domain of theCAR, provides the CAR with the ability to bind to the target antigen ofinterest. A binding domain can be any protein, polypeptide,oligopeptide, or peptide that possesses the ability to specificallyrecognize and bind to a biological molecule (e.g., a cell surfacereceptor or tumor protein, or a component thereof). A binding domainincludes any naturally occurring, synthetic, semi-synthetic, orrecombinantly produced binding partner for a biological molecule ofinterest. For example, and as further described herein, a binding domainmay be antibody light chain and heavy chain variable regions, or thelight and heavy chain variable regions can be joined together in asingle chain and in either orientation (e.g., VL-VH or VH-VL). A varietyof assays are known for identifying binding domains of the presentdisclosure that specifically bind with a particular target, includingWestern blot, ELISA, flow cytometry, or surface plasmon resonanceanalysis (e.g., using BIACORE analysis), and are described herein. Thetarget may be any antigen of clinical interest against which it would bedesirable to trigger an effector immune response that results in tumorkilling. In one embodiment, the target antigen of the binding domain ofthe chimeric antigen receptor is a conformational epitope of an HLA-A2presented HPV16E7 peptide on the surface of tumor cells, such as apeptide comprising amino acid residues 11-19 or 82-90 of HPV16E7.

Illustrative binding domains include antigen-binding proteins, such asantigen-binding fragments of an antibody, such as scFv, scTCR,extracellular domains of receptors, ligands for cell surfacemolecules/receptors, or receptor binding domains thereof, and tumorbinding proteins. In certain embodiments, the antigen-binding domainsincluded in a CAR of the invention can be a variable region (Fv), a CDR,a Fab, an scFv, a VH, a VL, a domain antibody variant (dAb), a camelidantibody (VHH), a fibronectin 3 domain variant, an ankyrin repeatvariant and other antigen-specific binding domain derived from otherprotein scaffolds.

In one embodiment, the binding domain of the CAR is ananti-HLA-A2:HPV16E7 single chain antibody (scFv), and may be a murine,human or humanized scFv. Single chain antibodies may be cloned form theV region genes of a hybridoma specific for a desired target. A techniquewhich can be used for cloning the variable region heavy chain (VH) andvariable region light chain (VL) has been described, for example, inOrlandi et al., PNAS, 1989; 86: 3833-3837. Thus, in certain embodiments,a binding domain comprises an antibody-derived binding domain but can bea non-antibody derived binding domain. An antibody-derived bindingdomain can be a fragment of an antibody or a genetically engineeredproduct of one or more fragments of the antibody, which fragment isinvolved in binding with the antigen.

In certain embodiments, the CARs of the present invention may comprise alinker between the various domains, added for appropriate spacing andconformation of the molecule. For example, in one embodiment, there maybe a linker between the binding domain VH or VL which may be between1-10 amino acids long. In other embodiments, the linker between any ofthe domains of the chimeric antigen receptor may be between 1-20 or 20amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. Infurther embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 amino acids long. Ranges including the numbers described hereinare also included herein, e.g., a linker 10-30 amino acids long.

In certain embodiments, linkers suitable for use in the CAR describedherein are flexible linkers. Suitable linkers can be readily selectedand can be of any of a suitable of different lengths, such as from 1amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 aminoacids, from 3 amino acids to 12 amino acids, including 4 amino acids to10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers, where n is an integer of at least one, glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers known inthe art. Glycine and glycine-serine polymers are relativelyunstructured, and therefore may be able to serve as a neutral tetherbetween domains of fusion proteins such as the CARs described herein.Glycine accesses significantly more phi-psi space than even alanine, andis much less restricted than residues with longer side chains (seeScheraga, Rev. Computational Chem. 11173-142 (1992)). The ordinarilyskilled artisan will recognize that design of a CAR can include linkersthat are all or partially flexible, such that the linker can include aflexible linker as well as one or more portions that confer lessflexible structure to provide for a desired CAR structure.

The binding domain of the CAR may be followed by a “spacer,” or,“hinge,” which refers to the region that moves the antigen-bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen-binding and activation (Patel et al., Gene Therapy,1999; 6: 412-419). The hinge region in a CAR is generally between thetransmembrane (TM) and the binding domain. In certain embodiments, ahinge region is an immunoglobulin hinge region and may be a wild typeimmunoglobulin hinge region or an altered wild type immunoglobulin hingeregion. Other exemplary hinge regions used in the CARs described hereininclude the hinge region derived from the extracellular regions of type1 membrane proteins such as CD8alpha, CD4, CD28 and CD7, which may bewild-type hinge regions from these molecules or may be altered. In oneembodiment, the hinge region comprises a CD8alpha hinge.

The “transmembrane,” region or domain is the portion of the CAR thatanchors the extracellular binding portion to the plasma membrane of theimmune effector cell, and facilitates binding of the binding domain tothe target antigen. The transmembrane domain may be a CD3zetatransmembrane domain, however other transmembrane domains that may beemployed include those obtained from CD8alpha, CD4, CD28, CD45, CD9,CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In oneembodiment, the transmembrane domain is the transmembrane domain ofCD137. In certain embodiments, the transmembrane domain is synthetic inwhich case it would comprise predominantly hydrophobic residues such asleucine and valine.

The “intracellular signaling domain,” refers to the part of the chimericantigen receptor protein that participates in transducing the message ofeffective CAR binding to a target antigen into the interior of theimmune effector cell to elicit effector cell function, e.g., activation,cytokine production, proliferation and cytotoxic activity, including therelease of cytotoxic factors to the CAR-bound target cell, or othercellular responses elicited with antigen-binding to the extracellularCAR domain. The term “effector function” refers to a specializedfunction of the cell. Effector function of the T cell, for example, maybe cytolytic activity or help or activity including the secretion of acytokine. Thus, the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces the effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal. The intracellular signaling domain is also known asthe, “signal transduction domain,” and is typically derived fromportions of the human CD3 or FcRy chains.

It is known that signals generated through the T cell receptor alone areinsufficient for full activation of the T cell and that a secondary, orcostimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequences: those that initiate antigen dependent primary activationthrough the T cell receptor (primary cytoplasmic signaling sequences)and those that act in an antigen independent manner to provide asecondary or costimulatory signal (secondary cytoplasmic signalingsequences). Primary cytoplasmic signaling sequences regulate primaryactivation of the T cell receptor complex either an inhibitory way, orin an inhibitory way. Primary cytoplasmic signaling sequences that actin a costimulatory manner may contain signaling motifs which are knownas immunoreceptor tyrosine-based activation motif or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular used in the invention include those derived fromTCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22,CD79a, CD79b and CD66d. In certain particular embodiments, theintracellular signaling domain of the anti-HLA-A2:HPV16E7 CARs describedherein are derived from CD3zeta or FcRgamma.

As used herein, the term, “co-stimulatory signaling domain,” or“co-stimulatory domain”, refers to the portion of the CAR comprising theintracellular domain of a co-stimulatory molecule. Co-stimulatorymolecules are cell surface molecules other than antigen receptors or Fcreceptors that provide a second signal required for efficient activationand function of T lymphocytes upon binding to antigen. Examples of suchco-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40(CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C,B7-H2 and a ligand that specifically binds CD83. Accordingly, while thepresent disclosure provides exemplary costimulatory domains derived fromCD3zeta and 4-1BB, other costimulatory domains are contemplated for usewith the CARs described herein. The inclusion of one or moreco-stimulatory signaling domains may enhance the efficacy and expansionof T cells expressing CAR receptors. The intracellular signaling andco-stimulatory signaling domains may be linked in any order in tandem tothe carboxyl terminus of the transmembrane domain.

Although scFv-based CARs engineered to contain a signaling domain fromCD3 or FcRgamma have been shown to deliver a potent signal for T cellactivation and effector function, they are not sufficient to elicitsignals that promote T cell survival and expansion in the absence of aconcomitant co-stimulatory signal. Other CARs containing a bindingdomain, a hinge, a transmembrane and the signaling domain derived fromCD3zeta or FcRgamma together with one or more co-stimulatory signalingdomains (e.g., intracellular co-stimulatory domains derived from CD28,CD137, CD134 and CD278) may more effectively direct antitumor activityas well as increased cytokine secretion, lytic activity, survival andproliferation in CAR expressing T cells in vitro, and in animal modelsand cancer patients (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenitoet al., PNAS, 2009; 106:3360-3365).

In one embodiment, the HLA-A2:HPV16E7 CARs of the invention comprise (a)an anti-HLA-A2:HPV16E7 scFv as a binding domain (e.g., an scFv havingbinding regions (e.g., CDRs or variable domains) from any one or more ofthe HLA-A2:HPV16E7 antibodies described in Table 1) (b) a hinge regionderived from human CD8alpha, (c) a human CD8alpha transmembrane domain,and (d) a human T cell receptor CD3 zeta chain (CD3) intracellularsignaling domain, and optionally one or more co-stimulatory signalingdomains derived from CD28, CD137, CD134, and CD278. In one embodiment,the different protein domains are arranged from amino to carboxylterminus in the following order: binding domain, hinge region andtransmembrane domain. The intracellular signaling domain and optionalco-stimulatory signaling domains are linked to the transmembrane carboxyterminus in any order in tandem to form a single chain chimericpolypeptide. In one embodiment, a nucleic acid construct encoding anHLA-A2:HPV16E7 CAR is a chimeric nucleic acid molecule comprising anucleic acid molecule comprising different coding sequences, forexample, (5′ to 3′) the coding sequences of a human anti-HLA-A2:HPV16E7scFv, a human CD8alpha-hinge, a human CD8alpha transmembrane domain anda CD3zeta intracellular signaling domain. In another embodiment, anucleic acid construct encoding an HLA-A2:HPV16E7 CAR is a chimericnucleic acid molecule comprising a nucleic acid molecule comprisingdifferent coding sequences, for example, (5′ to 3′) the coding sequencesof a human anti-HLA-A2:HPV16E7 scFv, a human CD8alpha-hinge, a humanCD8alpha transmembrane domain, a CD137 co-stimulatory domain, and aCD3zeta co-stimulatory domain. In certain embodiments, a nucleic acidconstruct encoding an HLA-A2:HPV16E7 CAR is a chimeric nucleic acidmolecule comprising a nucleic acid molecule comprising different codingsequences, for example, (5′ to 3′) the coding sequences of a humananti-HLA-A2:HPV16E7 scFv, a human CD8alpha-hinge, a human CD8alphatransmembrane domain, a CD137 co-stimulatory domain, and a CD3zetaco-stimulatory domain, wherein the anti-HLA-A2:HPV16E7 scFv comprises aV_(H) selected from the group consisting of SEQ ID Nos: 2, 34, 82, 194,282, and 506, and a V_(L) selected from the group consisting of SEQ IDNos: 10, 42, 90, 202, 290 and 514. In some embodiments, the presentinvention includes a nucleic acid molecule that encodes for aHLA-A2:HPV16E7 CAR selected from the group consisting of SEQ ID Nos:540, 541, 542, 543, 544 and 545.

In certain embodiments, the polynucleotide encoding the CAR describedherein is inserted into a vector. The term “vector” as used hereinrefers to a vehicle into which a polynucleotide encoding a protein maybe covalently inserted so as to bring about the expression of thatprotein and/or the cloning of the polynucleotide. Such vectors may alsobe referred to as “expression vectors”. The isolated polynucleotide maybe inserted into a vector using any suitable methods known in the art,for example, without limitation, the vector may be digested usingappropriate restriction enzymes and then may be ligated with theisolated polynucleotide having matching restriction ends. Expressionvectors have the ability to incorporate and express heterologous ormodified nucleic acid sequences coding for at least part of a geneproduct capable of being transcribed in a cell. In most cases, RNAmolecules are then translated into a protein. Expression vectors cancontain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are discussed infra. An expressionvector may comprise additional elements, for example, the expressionvector may have two replication systems, thus allowing it to bemaintained in two organisms, for example in human cells for expressionand in a prokaryotic host for cloning and amplification.

The expression vector may have the necessary 5′ upstream and 3′downstream regulatory elements such as promoter sequences such as CMV,PGK and EF1alpha. promoters, ribosome recognition and binding TATA box,and 3′ UTR AAUAAA transcription termination sequence for the efficientgene transcription and translation in its respective host cell. Othersuitable promoters include the constitutive promoter of simian virus 40(SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTRpromoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediateearly promoter, and rous sarcoma virus promoter. Human gene promotersmay also be used, including, but not limited to the actin promoter, themyosin promoter, the hemoglobin promoter, and the creatine kinasepromoter. In certain embodiments inducible promoters are alsocontemplated as part of the vectors expressing chimeric antigenreceptor. This provides a molecular switch capable of turning onexpression of the polynucleotide sequence of interest or turning offexpression. Examples of inducible promoters include, but are not limitedto a metallothionine promoter, a glucocorticoid promoter, a progesteronepromoter, or a tetracycline promoter.

The expression vector may have additional sequence such as 6×-histidine,c-Myc, and FLAG tags which are incorporated into the expressed CARs.Thus, the expression vector may be engineered to contain 5′ and 3′untranslated regulatory sequences that sometimes can function asenhancer sequences, promoter regions and/or terminator sequences thatcan facilitate or enhance efficient transcription of the nucleic acid(s)of interest carried on the expression vector. An expression vector mayalso be engineered for replication and/or expression functionality(e.g., transcription and translation) in a particular cell type, celllocation, or tissue type. Expression vectors may include a selectablemarker for maintenance of the vector in the host or recipient cell.

Examples of vectors are plasmid, autonomously replicating sequences, andtransposable elements. Additional exemplary vectors include, withoutlimitation, plasmids, phagemids, cosmids, artificial chromosomes such asyeast artificial chromosome (YAC), bacterial artificial chromosome(BAC), or P1-derived artificial chromosome (PAC), bacteriophages such aslambda phage or M13 phage, and animal viruses. Examples of categories ofanimal viruses useful as vectors include, without limitation, retrovirus(including lentivirus), adenovirus, adeno-associated virus, herpesvirus(e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, andpapovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™Bicistronic Expression System (Neo) vectors (Clontrch), pClneo vectors(Promega) for expression in mammalian cells; pLenti4/V5-DEST™,pLenti6/V5-DEST™, and pLenti6.2N5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells. Thecoding sequences of the CARs disclosed herein can be ligated into suchexpression vectors for the expression of the chimeric protein inmammalian cells.

In certain embodiments, the nucleic acids encoding the CAR of thepresent invention are provided in a viral vectors. A viral vector can bethose derived from retrovirus, lentivirus, or foamy virus. As usedherein, the term, “viral vector,” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the coding sequence for a the various chimeric proteinsdescribed herein in place of nonessential viral genes. The vector and/orparticle can be utilized for the purpose of transferring DNA, RNA orother nucleic acids into cells either in vitro or in vivo. Numerousforms of viral vectors are known in the art.

In certain embodiments, the viral vector containing the coding sequencefor a CAR described herein is a retroviral vector or a lentiviralvector. The term “retroviral vector” refers to a vector containingstructural and functional genetic elements that are primarily derivedfrom a retrovirus. The term “lentiviral vector” refers to a vectorcontaining structural and functional genetic elements outside the LTRsthat are primarily derived from a lentivirus.

The retroviral vectors for use herein can be derived from any knownretrovirus (e.g., type c retroviruses, such as Moloney murine sarcomavirus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammarytumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemiavirus (FLV), spumavirus, Friend, Murine Stem Cell Virus (MSCV) and RousSarcoma Virus (RSV)). Retroviruses” of the invention also include humanT cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family ofretroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2,simian immunodeficiency virus (SIV), feline immunodeficiency virus(FIV), equine immnodeficiency virus (EIV), and other classes ofretroviruses.

A lentiviral vector for use herein refers to a vector derived from alentivirus, a group (or genus) of retroviruses that give rise to slowlydeveloping disease. Viruses included within this group include HIV(human immunodeficiency virus; including HIV type 1, and HIV type 2);visna-maedi; a caprine arthritis-encephalitis virus; equine infectiousanemia virus; feline immunodeficiency virus (FIV); bovine immunedeficiency virus (BIV); and simian immunodeficiency virus (SIV).Preparation of the recombinant lentivirus can be achieved using themethods according to Dull et al. and Zufferey et al. (Dull et al., J.Virol., 1998; 72: 8463-8471 and Zufferey et al., J. Virol. 1998;72:9873-9880).

Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use inthe present invention can be formed using standard cloning techniques bycombining the desired DNA sequences in the order and orientationdescribed herein (Current Protocols in Molecular Biology, Ausubel, F. M.et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14and other standard laboratory manuals; Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573).

Suitable sources for obtaining retroviral (i.e., both lentiviral andnon-lentiviral) sequences for use in forming the vectors include, forexample, genomic RNA and cDNAs available from commercially availablesources, including the Type Culture Collection (ATCC), Rockville, Md.The sequences also can be synthesized chemically.

For expression of a HLA-A2:HPV16E7 CAR, the vector may be introducedinto a host cell to allow expression of the polypeptide within the hostcell. The expression vectors may contain a variety of elements forcontrolling expression, including without limitation, promotersequences, transcription initiation sequences, enhancer sequences,selectable markers, and signal sequences. These elements may be selectedas appropriate by a person of ordinary skill in the art, as describedabove. For example, the promoter sequences may be selected to promotethe transcription of the polynucleotide in the vector. Suitable promotersequences include, without limitation, T7 promoter, T3 promoter, SP6promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40promoter. Enhancer sequences may be selected to enhance thetranscription of the polynucleotide. Selectable markers may be selectedto allow selection of the host cells inserted with the vector from thosenot, for example, the selectable markers may be genes that conferantibiotic resistance. Signal sequences may be selected to allow theexpressed polypeptide to be transported outside of the host cell.

For cloning of the polynucleotide, the vector may be introduced into ahost cell (an isolated host cell) to allow replication of the vectoritself and thereby amplify the copies of the polynucleotide containedtherein. The cloning vectors may contain sequence components generallyinclude, without limitation, an origin of replication, promotersequences, transcription initiation sequences, enhancer sequences, andselectable markers. These elements may be selected as appropriate by aperson of ordinary skill in the art. For example, the origin ofreplication may be selected to promote autonomous replication of thevector in the host cell.

In certain embodiments, the present disclosure provides isolated hostcells containing the vectors provided herein. The host cells containingthe vector may be useful in expression or cloning of the polynucleotidecontained in the vector. Suitable host cells can include, withoutlimitation, prokaryotic cells, fungal cells, yeast cells, or highereukaryotic cells such as mammalian cells. Suitable prokaryotic cells forthis purpose include, without limitation, eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis, Pseudomonas such as P.aeruginosa, and Streptomyces.

The CARs of the present invention are introduced into a host cell usingtransfection and/or transduction techniques known in the art. As usedherein, the terms, “transfection,” and, “transduction,” refer to theprocesses by which an exogenous nucleic acid sequence is introduced intoa host cell. The nucleic acid may be integrated into the host cell DNAor may be maintained extrachromosomally. The nucleic acid may bemaintained transiently or a may be a stable introduction. Transfectionmay be accomplished by a variety of means known in the art including butnot limited to calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics. Transductionrefers to the delivery of a gene(s) using a viral or retroviral vectorby means of viral infection rather than by transfection. In certainembodiments, retroviral vectors are transduced by packaging the vectorsinto virions prior to contact with a cell. For example, a nucleic acidencoding an anti-HLA-A2:HPV16E7 CAR carried by a retroviral vector canbe transduced into a cell through infection and pro virus integration.

As used herein, the term “genetically engineered” or “geneticallymodified” refers to the addition of extra genetic material in the formof DNA or RNA into the total genetic material in a cell. The terms,“genetically modified cells,” “modified cells,” and, “redirected cells,”are used interchangeably.

In particular, the CAR of the present invention is introduced andexpressed in immune effector cells so as to redirect their specificityto a target antigen of interest, e.g., a conformational epitope of anHLA-A2 displayed HPV16E7 peptide, e.g., amino acid residues 11-19 or82-90.

The present invention provides methods for making the immune effectorcells which express the CAR as described herein. In one embodiment, themethod comprises transfecting or transducing immune effector cellsisolated from a subject, such as a subject having an HPV16E7-associatedisease or disorder, such that the immune effector cells express one ormore CAR as described herein. In certain embodiments, the immuneeffector cells are isolated from an individual and genetically modifiedwithout further manipulation in vitro. Such cells can then be directlyre-administered into the individual. In further embodiments, the immuneeffector cells are first activated and stimulated to proliferate invitro prior to being genetically modified to express a CAR. In thisregard, the immune effector cells may be cultured before or after beinggenetically modified (i.e., transduced or transfected to express a CARas described herein).

Prior to in vitro manipulation or genetic modification of the immuneeffector cells described herein, the source of cells may be obtainedfrom a subject. In particular, the immune effector cells for use withthe CARs as described herein comprise T cells. Such recombinant T cellsare referred to herein as “T-bodies.”

In one embodiment of the present invention, a T-body includes a CAR ofthe invention comprising an extracellular target-specific bindingdomain, a transmembrane domain, an intracellular signaling domain (suchas a signaling domain derived from CD3zeta or FcRgamma), and/or one ormore co-stimulatory signaling domains derived from a co-stimulatorymolecule, such as, but not limited to, CD28, CD137, CD134 or CD278. Inanother embodiment of the present invention, a T-body includes a CAR ofthe invention comprising an extracellular target-specific bindingdomain, a transmembrane domain, a hinge or spacer region between theextracellular binding domain and the transmembrane domain, anintracellular signaling domain (such as a signaling domain derived fromCD3zeta or FcRgamma), and/or one or more co-stimulatory signalingdomains derived from a co-stimulatory molecule. In yet anotherembodiment of the present invention, a T-body includes a T-bodyconstruct CAR comprising an extracellular target-specific bindingdomain, and a T cell receptor constant domain. The extracellulartarget-specific binding domain suitable for use in a T-body comprisingany of the CARs described herein may comprise a Fab, a Fab′, a (Fab′)2,an Fv, or a single chain Fv (scFv) of an antigen-binding protein of theinvention.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, lymph nodes tissue, cord blood,thymus issue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments, T cell canbe obtained from a unit of blood collected from the subject using anynumber of techniques known to the skilled person, such as FICOLLseparation. In one embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocyte, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing. In one embodimentof the invention, the cells are washed with PBS. In an alternativeembodiment, the washed solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. As would be appreciated bythose of ordinary skill in the art, a washing step may be accomplishedby methods known to those in the art, such as by using a semiautomatedflowthrough centrifuge. After washing, the cells may be resuspended in avariety of biocompatible buffers or other saline solution with orwithout buffer. In certain embodiments, the undesirable components ofthe apheresis sample may be removed in the cell directly resuspendedculture media.

In certain embodiments, T cells are isolated from peripheral bloodmononuclear cells (PBMCs) by lysing the red blood cells and depletingthe monocytes, for example, by centrifugation through a PERCOLL™gradient. A specific subpopulation of T cells, such as CD28+, CD4+,CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positiveor negative selection techniques. For example, enrichment of a T cellpopulation by negative selection can be accomplished with a combinationof antibodies directed to surface markers unique to the negativelyselected cells. One method for use herein is cell sorting and/orselection via negative magnetic immunoadherence or flow cytometry thatuses a cocktail of monoclonal antibodies directed to cell surfacemarkers present on the cells negatively selected. For example, to enrichfor CD4+ cells by negative selection, a monoclonal antibody cocktailtypically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, andCD8. Flow cytometry and cell sorting may also be used to isolate cellpopulations of interest for use in the present invention.

PBMC may be used directly for genetic modification with the CARs usingmethods as described herein. In certain embodiments, after isolation ofPBMC, T lymphocytes are further isolated and in certain embodiments,both cytotoxic and helper T lymphocytes can be sorted into naive,memory, and effector T cell subpopulations either before or aftergenetic modification and/or expansion. CD8+ cells can be obtained byusing standard methods. In some embodiments, CD8+ cells are furthersorted into naive, central memory, and effector cells by identifyingcell surface antigens that are associated with each of those types ofCD8+ cells. In embodiments, memory T cells are present in both CD62L+and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC are sortedinto CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8and anti-CD62L antibodies. In some embodiments, the expression ofphenotypic markers of central memory TCM include CD45RO, CD62L, CCR7,CD28, CD3, and CD127 and are negative for granzyme B. In someembodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells.In some embodiments, effector T cells are negative for CD62L, CCR7,CD28, and CD127, and positive for granzyme B and perforin. In someembodiments, naive CD8+ T lymphocytes are characterized by theexpression of phenotypic markers of naive T cells including CD62L, CCR7,CD28, CD3, CD 127, and CD45RA.

In certain embodiments, CD4+ T cells are further sorted intosubpopulations. For example, CD4+ T helper cells can be sorted intonaive, central memory, and effector cells by identifying cellpopulations that have cell surface antigens. CD4+ lymphocytes can beobtained by standard methods. In some embodiments, naive CD4+ Tlymphocytes are CD45RO−, CD45RA+, CD62L+CD4+ T cell. In someembodiments, central memory CD4+ cells are CD62L positive and CD45ROpositive. In some embodiments, effector CD4+ cells are CD62L and CD45ROnegative.

The immune effector cells, such as T cells, can be genetically modifiedfollowing isolation using known methods, or the immune effector cellscan be activated and expanded (or differentiated in the case ofprogenitors) in vitro prior to being genetically modified. In anotherembodiment, the immune effector cells, such as T cells, are geneticallymodified with the chimeric antigen receptors described herein (e.g.,transduced with a viral vector comprising a nucleic acid encoding a CAR)and then are activated and expanded in vitro. Methods for activating andexpanding T cells are known in the art and are described, for example,in U.S. Pat. Nos. 6,905,874; 6,867,041; 6,797,514; WO2012079000, US2016/0175358. Generally, such methods include contacting PBMC orisolated T cells with a stimulatory agent and costimulatory agent, suchas anti-CD3 and anti-CD28 antibodies, generally attached to a bead orother surface, in a culture medium with appropriate cytokines, such asIL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serveas a “surrogate” antigen presenting cell (APC). In other embodiments,the T cells may be activated and stimulated to proliferate with feedercells and appropriate antibodies and cytokines using methods such asthose described in U.S. Pat. Nos. 6,040,177; 5,827,642; andWO2012129514.

The invention provides a population of modified immune effector cellsfor the treatment of an HPV-associated disease or disorder, e.g.,cancer, the modified immune effector cells comprising an HLA-A2:HPV16E7CAR as disclosed herein.

CAR-expressing immune effector cells prepared as described herein can beutilized in methods and compositions for adoptive immunotherapy inaccordance with known techniques, or variations thereof that will beapparent to those skilled in the art based on the instant disclosure.See, e.g., US Patent Application Publication No. 2003/0170238 toGruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.

In some embodiments, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a treatment-effective amount.Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with human serum albumin.

A treatment-effective amount of cells in the composition is at least 2cells (for example, at least 1 CD8+ central memory T cell and at least 1CD4+ helper T cell subset) or is more typically greater than 10² cells,and up to 10⁶ up to and including 10⁸ or 10⁹ cells and can be more than10¹⁰ cells. The number of cells will depend upon the ultimate use forwhich the composition is intended as will the type of cells includedtherein.

The cells may be autologous or heterologous to the patient undergoingtherapy. If desired, the treatment may also include administration ofmitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g.,IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13,Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction ofthe immune response.

The CAR expressing immune effector cell populations of the presentinvention may be administered either alone, or as a pharmaceuticalcomposition in combination with diluents and/or with other componentssuch as IL-2 or other cytokines or cell populations. Briefly,pharmaceutical compositions of the present invention may comprise aCAR-expressing immune effector cell population, such as T cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

The anti-tumor immune response induced in a subject by administering CARexpressing T cells described herein using the methods described herein,or other methods known in the art, may include cellular immune responsesmediated by cytotoxic T cells capable of killing infected cells,regulatory T cells, and helper T cell responses. Humoral immuneresponses, mediated primarily by helper T cells capable of activating Bcells thus leading to antibody production, may also be induced. Avariety of techniques may be used for analyzing the type of immuneresponses induced by the compositions of the present invention, whichare well described in the art; e.g., Current Protocols in Immunology,Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, EthanM. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

Thus, the present invention provides for methods of treating anindividual diagnosed with or suspected of having, or at risk ofdeveloping, an HPV-associated disease or disorder, e.g.,HPV16E7-positive cancer, comprising administering the individual atherapeutically effective amount of the CAR-expressing immune effectorcells as described herein.

In one embodiment, the invention provides a method of treating a subjectdiagnosed with an HPV16E7-positive cancer comprising removing immuneeffector cells from a subject diagnosed with an HPV16E7-positive cancer,genetically modifying said immune effector cells with a vectorcomprising a nucleic acid encoding a chimeric antigen receptor of theinstant invention, thereby producing a population of modified immuneeffector cells, and administering the population of modified immuneeffector cells to the same subject. In one embodiment, the immuneeffector cells comprise T cells.

The methods for administering the cell compositions described hereinincludes any method which is effective to result in reintroduction of exvivo genetically modified immune effector cells that either directlyexpress a CAR of the invention in the subject or on reintroduction ofthe genetically modified progenitors of immune effector cells that onintroduction into a subject differentiate into mature immune effectorcells that express the CAR. One method comprises transducing peripheralblood T cells ex vivo with a nucleic acid construct in accordance withthe invention and returning the transduced cells into the subject.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-HLA-A2:HPV16E7 antigen-binding proteins, e.g., antibodies, orantigen-biding fragments thereof, or CARs, of the present invention.Therapeutic compositions in accordance with the invention will beadministered with suitable carriers, excipients, and other agents thatare incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of the antigen-binding protein, e.g., antibody, orantigen-biding fragments thereof, may vary depending upon the age andthe size of a subject to be administered, target disease, conditions,route of administration, and the like. When an antigen-binding proteinof the present invention is used for treating a disease or disorder inan adult patient, or for preventing such a disease, it is advantageousto administer the antigen-binding protein, e.g., antibody, orantigen-biding fragments thereof, of the present invention normally at asingle dose of about 0.1 to about 60 mg/kg body weight, more preferablyabout 5 to about 60, about 20 to about 50, about 10 to about 50, about 1to about 10, or about 0.8 to about 11 mg/kg body weight. Depending onthe severity of the condition, the frequency and the duration of thetreatment can be adjusted. In certain embodiments, the antigen-bindingprotein, e.g., antibody, or antigen-biding fragments thereof, of theinvention can be administered as an initial dose of at least about 0.1mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, orabout 10 to about 200 mg, to about 100 mg, or to about 50 mg. In certainembodiments, the initial dose may be followed by administration of asecond or a plurality of subsequent doses of the antigen-bindingprotein, e.g., antibody, or antigen-biding fragments thereof, in anamount that can be approximately the same or less than that of theinitial dose, wherein the subsequent doses are separated by at least 1day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; atleast 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; atleast 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks;or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing mutant viruses, receptor mediated endocytosis (see, e.g., Wuet al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the antigen-binding proteins, e.g.,antibody, or antigen-biding fragments thereof, of the present inventionis also contemplated herein. Antigen binding protein-conjugatednanoparticles may be used both for therapeutic and diagnosticapplications. Antigen binding protein-conjugated nanoparticles andmethods of preparation and use are described in detail by Arruebo, M.,et al. 2009 (“Antibody-conjugated nanoparticles for biomedicalapplications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages,doi: 10.1155/2009/439389), incorporated herein by reference.Nanoparticles may be developed and conjugated to antigen-bindingproteins contained in pharmaceutical compositions to target tumor cellsor autoimmune tissue cells or virally infected cells. Nanoparticles fordrug delivery have also been described in, for example, U.S. Pat. No.8,257,740, or U.S. Pat. No. 8,246,995, each incorporated herein in itsentirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous, intracranial, intraperitoneal andintramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by methods publicly known. For example, theinjectable preparations may be prepared, e.g., by dissolving, suspendingor emulsifying the antigen-binding protein or its salt described abovein a sterile aqueous medium or an oily medium conventionally used forinjections. As the aqueous medium for injections, there are, forexample, physiological saline, an isotonic solution containing glucoseand other auxiliary agents, etc., which may be used in combination withan appropriate solubilizing agent such as an alcohol (e.g., ethanol), apolyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionicsurfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)], etc. As the oily medium, there areemployed, e.g., sesame oil, soybean oil, etc., which may be used incombination with a solubilizing agent such as benzyl benzoate, benzylalcohol, etc. The injection thus prepared is preferably filled in anappropriate ampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antigen-bindingprotein contained is generally about 5 to about 500 mg per dosage formin a unit dose; especially in the form of injection, it is preferredthat the antigen-binding protein is contained in about 5 to about 100 mgand in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Proteins

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by HPV16. For example, the present inventionprovides methods for treating a HPV-associated disease or disorder, suchas an HPV-associated cancer (e.g., a HPV16E7-positive cancer) (tumorgrowth inhibition) and/or HPV infections by administering ananti-HLA-A2:HPV16E7 antigen-binding protein (or pharmaceuticalcomposition comprising an anti-HLA-A2:HPV16E7 antigen-binding protein)as described herein to a patient in need of such treatment, andanti-HLA-A2:HPV16E7 antigen-binding proteins (or pharmaceuticalcomposition comprising an anti-HLA-A2:HPV16E7 antigen-binding protein)for use in the treatment of a HPV-associated cancer (tumor growthinhibition) and/or HPV infections. The antigen-binding proteins of thepresent invention are useful for the treatment, prevention, and/oramelioration of disease or disorder or condition such as anHPV-associated cancer or a HPV infection and/or for ameliorating atleast one symptom associated with such disease, disorder or condition.In the context of the methods of treatment described herein, theanti-HLA-A2:HPV16E7 antigen-binding protein may be administered as amonotherapy (i.e., as the only therapeutic agent) or in combination withone or more additional therapeutic agents (examples of which aredescribed elsewhere herein).

In some embodiments of the invention, the antibodies described hereinare useful for treating subjects suffering from primary or recurrentcancer, including, but not limited to, HPV-associated cancer, e.g.,squamous cell carcinomas, such as squamous cell carcinoma of head andneck, cervical cancer, anogenital cancer, oropharyngeal cancer.

The antigen-binding proteins may be used to treat early stage orlate-stage symptoms of the HPV-associated cancer. In one embodiment, anantibody or fragment thereof of the invention may be used to treatadvanced or metastatic cancer. The antigen-binding proteins are usefulin reducing or inhibiting or shrinking tumor growth. In certainembodiments, treatment with an antigen-binding protein of the inventionleads to more than 40% regression, more than 50% regression, more than60% regression, more than 70% regression, more than 80% regression ormore than 90% regression of a tumor in a subject. In certainembodiments, the antigen-binding proteins may be used to prevent relapseof a tumor. In certain embodiments, the antigen-binding proteins areuseful in extending progression-free survival or overall survival in asubject with HPV-associated cancer. In some embodiments, the antibodiesare useful in reducing toxicity due to chemotherapy or radiotherapywhile maintaining long-term survival in a patient suffering fromHPV-associated cancer.

In certain embodiments, the antigen-binding proteins of the inventionare useful to treat subjects suffering from a chronic HPV infection. Insome embodiments, the antigen-binding proteins of the invention areuseful in decreasing viral titers in the host.

One or more antibodies of the present invention may be administered torelieve or prevent or decrease the severity of one or more of thesymptoms or conditions of the disease or disorder.

It is also contemplated herein to use one or more antibodies of thepresent invention prophylactically to patients at risk for developing adisease or disorder such as HPV-associated disease or disorder, such asan HPV-associated cancer, and HPV infection.

In a further embodiment of the invention, the present antibodies areused for the preparation of a pharmaceutical composition for treatingpatients suffering from HPV-associated disease or disorder, such as anHPV-associated cancer, or HPV infection. In another embodiment of theinvention, the present antibodies are used as adjunct therapy with anyother agent or any other therapy known to those skilled in the artuseful for treating HPV-associated cancer or HPV infection.

Combination Therapies and Formulations

Combination therapies may include an anti-HLA-A2:HPV16E7 antigen-bindingprotein of the invention, such as a CAR of the invention (e.g., animmune effector cell comprising a CAR of the invention) or apharmaceutical composition of the invention, and any additionaltherapeutic agent that may be advantageously combined with anantigen-binding protein of the invention. The antigen-binding proteinsof the present invention may be combined synergistically with one ormore anti-cancer drugs or therapy used to treat or inhibit anHPV16E7-associated disease or disorder, such as HPV-positive cancer,e.g., squamous cell carcinoma, cervical cancer, anogenital cancer, headand neck cancer, or oropharyngeal cancer.

It is contemplated herein to use the anti-HLA-A2:HPV16E7 antigen-bindingproteins of the invention in combination with immunostimulatory and/orimmunosupportive therapies to inhibit tumor growth, and/or enhancesurvival of cancer patients. The immunostimulatory therapies includedirect immunostimulatory therapies to augment immune cell activity byeither “releasing the brake” on suppressed immune cells or “stepping onthe gas” to activate an immune response. Examples include targetingother checkpoint receptors, vaccination and adjuvants. Theimmunosupportive modalities may increase antigenicity of the tumor bypromoting immunogenic cell death, inflammation or have other indirecteffects that promote an anti-tumor immune response. Examples includeradiation, chemotherapy, anti-angiogenic agents, and surgery.

In various embodiments, one or more antigen-binding proteins of thepresent invention may be used in combination with a PD-1 inhibitor(e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab,pidilizumab, BGB-A317 or REGN2810), a PD-L1 inhibitor (e.g., ananti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab,MDX-1105, or REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITRinhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g.,an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), anindoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelialgrowth factor (VEGF) antagonist [e.g., a “VEGF-Trap” such as afliberceptor other VEGF-inhibiting fusion protein as set forth in U.S. Pat. No.7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof(e.g., bevacizumab, or ranibizumab) or a small molecule kinase inhibitorof VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2inhibitor (e.g., nesvacumab), a transforming growth factor beta (TGFβ)inhibitor, an epidermal growth factor receptor (EGFR) inhibitor (e.g.,erlotinib, cetuximab), a CD20 inhibitor (e.g., an anti-CD20 antibodysuch as rituximab), an antibody to a tumor-specific antigen [e.g., CA9,CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen(CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1,MART-1, and CA19-9], a vaccine (e.g., Bacillus Calmette-Guerin, a cancervaccine), an adjuvant to increase antigen presentation (e.g.,granulocyte-macrophage colony-stimulating factor), a bispecific antibody(e.g., CD3×CD20 bispecific antibody, or PSMA×CD3 bispecific antibody), acytotoxin, a chemotherapeutic agent (e.g., dacarbazine, temozolomide,cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin,carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin,paclitaxel, and vincristine), cyclophosphamide, radiotherapy, surgery,an IL-6R inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g.,dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21,and IL-15, an antibody-drug conjugate (ADC) (e.g., anti-CD19-DM4 ADC,and anti-DS6-DM4 ADC), an anti-inflammatory drug (e.g., corticosteroids,and non-steroidal anti-inflammatory drugs), a dietary supplement such asantioxidants or any other therapy care to treat cancer. In certainembodiments, the anti-HLA-A2:HPV16E7 antigen-binding protein of thepresent invention may be used in combination with an HPV vaccine.Exemplary HPV vaccines include Gardasil, Gardasil 9, and Cervarix,Lm-LLo-E7 (ADXS11-001; ADXS-HPV; Advaxis, Inc.); GLBL101c (GENOLAC BLCorp); TA-HPV (European Organization for Research and Treatment ofCancer (EORTC)); TG4001 (Transgene/Roche); MVA E2 (Instituto Mexicanodel Seguro Social); HPV16-SLP (ISA Pharmaceuticals); GL-0810 (GliknikInc.); Pepcan+Candin (University of Arkansas); GTL001 (ProCervix;Genticel); TA-CIN (Xenova Research Limited); TA-CIN+TA-HPV (CelticPharma); pNGVL4a-sig/E7(detox)/HSP70+TA-HPV (Sidney Kimmel ComprehensiveCancer Center); pNGVL4a-CRT/E7(detox) (Sidney Kimmel ComprehensiveCancer Center); GX-188E (Genexine, Inc); VGX-3100 (InovioPharmaceuticals); Dendritic Cells pulsed with HPV-16 and HPV-18 E7 andkeyhole limpet hemocyanin (National Institutes of Health); DC pulsedwith HPV+ tumor lysate (Department of Biotechnology (DBT, Govt. ofIndia)); PDS0101 (PDS Biotechnology Corp); ProCervix (Genticel); GX-188E(Genexine, Inc); pNGVL4a-CRT/E7(detox) (Sidney Kimmel ComprehensiveCancer Center); pNGVL4a-sig/E7(detox)/HSP70+TA-HPV (Sidney KimmelComprehensive Cancer Center); TVGV-1+GPI-0100 (THEVAX Genetics VaccineCo.); Pepcan+Candin (University of Arkansas); ISA101 (SLP-HPV-01;HPV16-SLP; ISA Pharmaceuticals); ADXS11-001 (Lm-LLo-E7; Advaxis, Inc.);ISA101 (SLP-HPV-01; HPV16-SLP; ISA Pharmaceuticals); DPX-E7 (Dana-FarberCancer Institute); ADXS11-001 (Lm-LLo-E7; Advaxis, Inc.); INO-3112(VGX-3100+INO-9012; Inovio Pharmaceuticals); ADXS11-001 (Lm-LLo-E7;Advaxis, Inc.); INO-3112 (VGX-3100+INO-9012; Inovio Pharmaceuticals);ISA101 (SLP-HPV-01; HPV16-SLP; ISA Pharmaceuticals); and TA-CIN+GPI-0100(Sidney Kimmel Comprehensive Cancer Center). In certain embodiments, theanti-HLA-A2:HPV16E7 antigen-binding protein of the present invention maybe used in combination with cancer vaccines including dendritic cellvaccines, oncolytic viruses, tumor cell vaccines, etc. to augment theanti-tumor response. Examples of cancer vaccines that can be used incombination with anti-HLA-A2:HPV16E7 antigen-binding proteins of thepresent invention include MAGE3 vaccine for melanoma and bladder cancer,MUC1 vaccine for breast cancer, EGFRv3 (e.g., Rindopepimut) for braincancer (including glioblastoma multiforme), or ALVAC-CEA (forCEA+cancers).

In certain embodiments, the anti-HLA-A2:HPV16E7 antigen-binding proteinsof the invention may be administered in combination with radiationtherapy in methods to generate long-term durable anti-tumor responsesand/or enhance survival of patients with cancer. In some embodiments,the anti-HLA-A2:HPV16E7 antigen-binding proteins of the invention may beadministered prior to, concomitantly or after administering radiationtherapy to a cancer patient. For example, radiation therapy may beadministered in one or more doses to tumor lesions followed byadministration of one or more doses of anti-HLA-A2:HPV16E7antigen-binding proteins of the invention. In some embodiments,radiation therapy may be administered locally to a tumor lesion toenhance the local immunogenicity of a patient's tumor (adjuvinatingradiation) and/or to kill tumor cells (ablative radiation) followed bysystemic administration of an anti-HLA-A2:HPV16E7 antigen-bindingprotein of the invention. For example, intracranial radiation may beadministered to a patient with brain cancer (e.g., glioblastomamultiforme) in combination with systemic administration of ananti-HLA-A2:HPV16E7 antigen-binding protein of the invention. In certainembodiments, the anti-HLA-A2:HPV16E7 antigen-binding proteins of theinvention may be administered in combination with radiation therapy anda chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist(e.g., aflibercept).

In certain embodiments, the anti-HLA-A2:HPV16E7 antigen-binding proteinsof the invention may be administered in combination with one or moreanti-viral drugs to treat chronic HPV infection. Examples of anti-viraldrugs include, but are not limited to, zidovudine, lamivudine, abacavir,ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine andcorticosteroids.

The additional therapeutically active agent(s)/component(s) may beadministered prior to, concurrent with, or after the administration ofthe anti-HLA-A2:HPV16E7 antigen-binding proteins of the presentinvention. For purposes of the present disclosure, such administrationregimens are considered the administration of an anti-HLA-A2:HPV16E7antigen-binding protein “in combination with” a second therapeuticallyactive component.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-HLA-A2:HPV16E7antigen-binding protein of the present invention. For example, a firstcomponent may be deemed to be administered “prior to” a second componentif the first component is administered 1 week before, 72 hours before,60 hours before, 48 hours before, 36 hours before, 24 hours before, 12hours before, 6 hours before, 5 hours before, 4 hours before, 3 hoursbefore, 2 hours before, 1 hour before, 30 minutes before, 15 minutesbefore, 10 minutes before, 5 minutes before, or less than 1 minutebefore administration of the second component. In other embodiments, theadditional therapeutically active component(s) may be administered to asubject after administration of an anti-HLA-A2:HPV16E7 antigen-bindingprotein of the present invention. For example, a first component may bedeemed to be administered “after” a second component if the firstcomponent is administered 1 minute after, 5 minutes after, 10 minutesafter, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after,3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hoursafter, 24 hours after, 36 hours after, 48 hours after, 60 hours after,72 hours after administration of the second component. In yet otherembodiments, the additional therapeutically active component(s) may beadministered to a subject concurrent with administration of ananti-HLA-A2:HPV16E7 antigen-binding protein of the present invention.“Concurrent” administration, for purposes of the present invention,includes, e.g., administration of an anti-HLA-A2:HPV16E7 antigen-bindingprotein and an additional therapeutically active component to a subjectin a single dosage form (e.g., co-formulated), or in separate dosageforms administered to the subject within about 30 minutes or less ofeach other. If administered in separate dosage forms, each dosage formmay be administered via the same route (e.g., both theanti-HLA-A2:HPV16E7 antigen-binding protein and the additionaltherapeutically active component may be administered intravenously,subcutaneously, etc.); alternatively, each dosage form may beadministered via a different route (e.g., the anti-HLA-A2:HPV16E7antigen-binding protein may be administered intravenously, and theadditional therapeutically active component may be administeredsubcutaneously). In any event, administering the components in a singledosage from, in separate dosage forms by the same route, or in separatedosage forms by different routes are all considered “concurrentadministration,” for purposes of the present disclosure. For purposes ofthe present disclosure, administration of an anti-HLA-A2:HPV16E7antigen-binding protein “prior to”, “concurrent with,” or “after” (asthose terms are defined herein above) administration of an additionaltherapeutically active component is considered administration of ananti-HLA-A2:HPV16E7 antigen-binding protein “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which ananti-HLA-A2:HPV16E7 antigen-binding protein of the present invention isco-formulated with one or more of the additional therapeutically activecomponent(s) as described elsewhere herein using a variety of dosagecombinations.

Administrative Regimens

According to certain embodiments of the present invention, multipledoses of an anti-HLA-A2:HPV16E7 antigen-binding protein (or apharmaceutical composition comprising a combination of ananti-HLA-A2:HPV16E7 antigen-binding protein and any of the additionaltherapeutically active agents mentioned herein) may be administered to asubject over a defined time course. The methods according to this aspectof the invention comprise sequentially administering to a subjectmultiple doses of an anti-HLA-A2:HPV16E7 antigen-binding protein of theinvention. As used herein, “sequentially administering” means that eachdose of anti-HLA-A2:HPV16E7 antigen-binding protein is administered tothe subject at a different point in time, e.g., on different daysseparated by a predetermined interval (e.g., hours, days, weeks ormonths). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of ananti-HLA-A2:HPV16E7 antigen-binding protein, followed by one or moresecondary doses of the anti-HLA-A2:HPV16E7 antigen-binding protein, andoptionally followed by one or more tertiary doses of theanti-HLA-A2:HPV16E7 antigen-binding protein. The anti-HLA-A2:HPV16E7antigen-binding protein may be administered at a dose between 0.1 mg/kgto 100 mg/kg body weight of the subject.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-HLA-A2:HPV16E7antigen-binding protein of the invention. Thus, the “initial dose” isthe dose which is administered at the beginning of the treatment regimen(also referred to as the “baseline dose”); the “secondary doses” are thedoses which are administered after the initial dose; and the “tertiarydoses” are the doses which are administered after the secondary doses.The initial, secondary, and tertiary doses may all contain the sameamount of anti-HLA-A2:HPV16E7 antigen-binding protein, but generally maydiffer from one another in terms of frequency of administration. Incertain embodiments, however, the amount of anti-HLA-A2:HPV16E7antigen-binding protein contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain embodiments, the amount of anti-HLA-A2:HPV16E7antigen-binding protein contained in the initial, secondary and/ortertiary doses may be sub-optimal or sub-therapeutic. As used herein,the terms “sub-therapeutic” or “sub-optimal” refer to an antibody doseadministered at too low a level to produce a therapeutic effect or belowthe level necessary to treat a disease such as cancer.

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-HLA-A2:HPV16E7 antigen-binding proteinwhich is administered to a patient prior to the administration of thevery next dose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-HLA-A2:HPV16E7 antigen-binding protein. For example, incertain embodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antigen Binding Proteins

The anti-HLA-A2:HPV16E7 antigen-binding proteins of the presentinvention may be used to detect and/or measure HPV16E7 in a sample,e.g., for diagnostic purposes. Some embodiments contemplate the use ofone or more antigen-binding proteins of the present invention in assaysto detect a disease or disorder such as HPV-associated disease ordisorder, such as an HPV16E7-positive cancer, or HPV infection.Exemplary diagnostic assays for HPV16E7 may comprise, e.g., contacting asample, obtained from a subject (e.g., a patient), with ananti-HLA-A2:HPV16E7 antigen-binding protein of the invention, whereinthe anti-HLA-A2:HPV16E7 antigen-binding protein is labeled with adetectable label or reporter molecule or used as a capture ligand toselectively isolate HPV16E7 from subject samples. Alternatively, anunlabeled anti-HLA-A2:HPV16E7 antigen-binding protein can be used indiagnostic applications in combination with a secondary antigen-bindingprotein, e.g., antibody, which is itself detectably labeled. Thedetectable label or reporter molecule can be a radioisotope, such as ³H,¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure HPV16E7in a sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in HPV16E7 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from asubject, which contains detectable quantities of either HPV16E7 protein,or fragments thereof, under normal or pathological conditions.Generally, levels of HPV16E7 in a particular sample obtained from ahealthy patient (e.g., a patient not afflicted with a HPV16E7-associateddisease or disorder, e.g., HPV16E7-positive cancer) will be measured toinitially establish a baseline, or standard, level of HPV16E7. Thisbaseline level of HPV16E7 can then be compared against the levels ofHPV16E7 measured in samples obtained from individuals suspected ofhaving a cancer-related condition, or symptoms associated with suchcondition.

The antigen-binding proteins specific for HPV16E7 may contain noadditional labels or moieties, or they may contain an N-terminal orC-terminal label or moiety. In one embodiment, the label or moiety isbiotin. In a binding assay, the location of a label (if any) maydetermine the orientation of the peptide relative to the surface uponwhich the peptide is bound. For example, if a surface is coated withavidin, a peptide containing an N-terminal biotin will be oriented suchthat the C-terminal portion of the peptide will be distal to thesurface.

Aspects of the invention relate to use of the disclosed antigen-bindingproteins as markers for predicting prognosis of HPV16E7-positive canceror HPV infection in patients. Antigen binding proteins of the presentinvention may be used in diagnostic assays to evaluate prognosis ofcancer in a patient and to predict survival.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: Generation of Human Antibodies to HLA-A2:HPV16E7

Human antibodies to HLA-A2:HPV16E7 were generated using peptidefragments of HPV16E7 that include either amino acids 11-19 (YMLDLQPET;SEQ ID NO: 538) of GenBank Accession NP_041326.1 (SEQ ID NO: 537) oramino acid residues 82-90 (LLMGTLGIV; SEQ ID NO: 539) of GenBankAccession NP_041326.1 (SEQ ID NO: 537), coupled to HLA-A2. The immunogenwas administered directly, with an adjuvant to stimulate the immuneresponse, to a VELOCIMMUNE® mouse (i.e., an engineered mouse comprisingDNA encoding human Immunoglobulin heavy and kappa light chain variableregions), e.g., as described in U.S. Pat. No. 8,502,018. The antibodyimmune response was monitored by an HLA-A2:HPV16E7-specific immunoassay.When a desired immune response was achieved splenocytes were harvestedand fused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce HLA-A2:HPV16E7-specificantibodies. Using this technique, and the immunogen described above,several anti-HPV16E7 chimeric antibodies (i.e., antibodies possessinghuman variable domains and mouse constant domains) were obtained.Exemplary antibodies generated in this manner were designated asfollows: H4sH17364N; H4sH17368N2; H4sH17930N; H4sH17930N2; H4sH17363Nand H4sH17368N3.

Anti-HLA-A2:HPV16E7 antibodies were also isolated directly fromantigen-positive B cells (from either of the immunized mice) withoutfusion to myeloma cells, as described in U.S. Pat. No. 7,582,298, hereinspecifically incorporated by reference in its entirety. Using thismethod, several fully human anti-HLA-A2:HPV16E7 antibodies (i.e.,antibodies possessing human variable domains and human constant domains)were obtained.

Exemplary antibodies generated according to the foregoing methods weredesignated as follows: H4sH17670P; H4sH17672P; H4sH17673P; H4sH17675P;H4sH17680P; H4sH17697P; H4sH17707P; H4sH17715P; H4sH17726P; H4sH17730P;H4sH21051P; H4sH21054P; H4sH21055P; H4sH21058P; H4sH21064P; H4sH21073P;H4sH21077P; H4sH21079P; H4sH21080P; H4sH21083P; H4sH21086P; H4sH21090P;H4sH21091P; H4sH21093P; H4sH21099P; H4sH21100P; H4sH21103P; andH4sH21104P.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid andNucleotide Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-HLA-A2:HPV16E7antibodies of the invention. The corresponding nucleic acid sequenceidentifiers are set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4sH17364N 2 4 6 8 10 1214 16 H4sH17368N2 18 20 22 24 26 28 30 32 H4sH17670P 34 36 38 40 42 4446 48 H4sH17672P 50 52 54 56 58 60 62 64 H4sH17673P 66 68 70 72 74 76 7880 H4sH17675P 82 84 86 88 90 92 94 96 H4sH17680P 98 100 102 104 106 108110 112 H4sH17697P 114 116 118 120 122 124 126 128 H4sH17707P 130 132134 136 138 140 142 144 H4sH17715P 146 148 150 152 154 156 158 160H4sH17726P 162 164 166 168 170 172 174 176 H4sH17730P 178 180 182 184186 188 190 192 H4sH17930N 210 212 214 216 202 204 206 208 H4sH17930N2194 196 198 200 202 204 206 208 H4sH21051P 218 220 222 224 226 228 230232 H4sH21054P 234 236 238 240 242 244 246 248 H4sH21055P 250 252 254256 258 260 262 264 H4sH21058P 266 268 270 272 274 276 278 280H4sH21064P 282 284 286 288 290 292 294 296 H4sH21073P 298 300 302 304306 308 310 312 H4sH21077P 314 316 318 320 322 324 326 328 H4sH21079P330 332 334 336 338 340 342 344 H4sH21080P 346 348 350 352 354 356 358360 H4sH21083P 362 364 366 368 370 372 374 376 H4sH21086P 378 380 382384 386 388 390 392 H4sH21090P 394 396 398 400 402 404 406 408H4sH21091P 410 412 414 416 418 420 422 424 H4sH21093P 426 428 430 432434 436 438 440 H4sH21099P 442 444 446 448 450 452 454 456 H4sH21100P458 460 462 464 466 468 470 472 H4sH21103P 474 476 478 480 482 484 486488 H4sH21104P 490 492 494 496 498 500 502 504 H4sH17363N 506 508 510512 514 516 518 520 H4sH17368N3 522 524 526 528 530 532 534 536

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4sH17364N 1 35 7 9 11 13 15 H4sH17368N2 17 19 21 23 25 27 29 31 H4sH17670P 33 35 3739 41 43 45 47 H4sH17672P 49 51 53 55 57 59 61 63 H4sH17673P 65 67 69 7173 75 77 79 H4sH17675P 81 83 85 87 89 91 93 95 H4sH17680P 97 99 101 103105 107 109 111 H4sH17697P 113 115 117 119 121 123 125 127 H4sH17707P129 131 133 135 137 139 141 143 H4sH17715P 145 147 149 151 153 155 157159 H4sH17726P 161 163 165 167 169 171 173 175 H4sH17730P 177 179 181183 185 187 189 191 H4sH17930N 209 211 213 215 201 203 205 207H4sH17930N2 193 195 197 199 201 203 205 207 H4sH21051P 217 219 221 223225 227 229 231 H4sH21054P 233 235 237 239 241 243 245 247 H4sH21055P249 251 253 255 257 259 261 263 H4sH21058P 265 267 269 271 273 275 277279 H4sH21064P 281 283 285 287 289 291 293 295 H4sH21073P 297 299 301303 305 307 309 311 H4sH21077P 313 315 317 319 321 323 325 327H4sH21079P 329 331 333 335 337 339 341 343 H4sH21080P 345 347 349 351353 355 357 359 H4sH21083P 361 363 365 367 369 371 373 375 H4sH21086P377 379 381 383 385 387 389 391 H4sH21090P 393 395 397 399 401 403 405407 H4sH21091P 409 411 413 415 417 419 421 423 H4sH21093P 425 427 429431 433 435 437 439 H4sH21099P 441 443 445 447 449 451 453 455H4sH21100P 457 459 461 463 465 467 469 471 H4sH21103P 473 475 477 479481 483 485 487 H4sH21104P 489 491 493 495 497 499 501 503 H4sH17363N505 507 509 511 513 515 517 519 H4sH17368N3 521 523 525 527 529 531 533535

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” “H4sH,” “H4H,” etc.), followed by anumerical identifier (e.g. “17670,” “17930,” etc., as shown in Table 1),followed by a “P,” “N,” or “N2” suffix. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H4sH17670P,” “H4sH17930N,” “H4sH17368N2,” etc. The H4sH and H4Hprefixes on the antibody designations used herein indicate theparticular Fc region isotype of the antibody. For example, an “H4sH”antibody has a human IgG4 Fc with 2 or more amino acid changes asdisclosed in U.S. Patent Publication No. 20140243504 (hereinincorporated in its entirety), an “H4H” antibody has a human IgG4 Fcwith a serine to proline mutation in the hinge region (S108P), an “H1M”antibody has a mouse IgG1 Fc, and an “H2M” antibody has a mouse IgG2 Fc(all variable regions are fully human as denoted by the first ‘H’ in theantibody designation). As will be appreciated by a person of ordinaryskill in the art, an antibody having a particular Fc isotype can beconverted to an antibody with a different Fc isotype (e.g., an antibodywith a mouse IgG1 Fc can be converted to an antibody with a human IgG4,etc.), but in any event, the variable domains (including the CDRs)—whichare indicated by the numerical identifiers shown in Table 1—will remainthe same, and the binding properties to antigen are expected to beidentical or substantially similar regardless of the nature of the Fcdomain.

In certain embodiments, selected antibodies with a mouse IgG1 Fc wereconverted to antibodies with human IgG4 Fc. In certain embodiments, theantibody comprises a human IgG4 Fc with 2 or more amino acid changes asdisclosed in U.S. Patent Publication No. 20100331527 (hereinincorporated in its entirety). In one embodiment, the IgG4 Fc domaincomprises a serine to proline mutation in the hinge region (S108P) topromote dimer stabilization.

Table 3 sets forth the amino acid sequence identifiers of heavy chainand light chain sequences of selected antibodies of the invention.

TABLE 3 Heavy chain and light chain sequence identifiers Antibody SEQ IDNOs: Designation Heavy Chain Light Chain H4sH17363N 578 579 H4sH17364N580 581 H4sH17670P 582 583 H4sH17675P 584 585 H4sH17930N2 586 587H4sH21058P 588 589 H4sH21064P 590 591 H4sH21104P 592 593

Example 3: Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each heavy chain variableregion (HCVR) and light chain variable region (LCVR) (Table 4).

TABLE 4 Antibody HCVR (HPV) LCVR (HPV) Designation V_(H) D_(H) J_(H)V_(H) J_(H) H4sH17363N V3-23 D6-6 J6 V1-39 J5 H4sH17364N V3-23 D6-6 J6V1-39 J5 H4sH17368N2 V3-23 D3-9 J4 V1-39 J5 H4sH17368N3 V3-23 D3-9 J4V1-39 J5 H4sH17670P V3-64 D1-26 J6 V1-39 J5 H4sH17672P V3-64 D1-26 J6V1-39 J5 H4sH17673P V3-23 D4-11 J6 V1-39 J5 H4sH17675P V3-64 D1-26 J6V1-39 J5 H4sH17680P V3-23 D4-23 J6 V1-39 J5 H4sH17697P V3-11 D6-13 J4V1-39 J2 H4sH17707P V3-23 D1-20 J4 V1-39 J5 H4sH17715P V6-1 D1-7 J3V1-39 J2 H4sH17726P V1-18 D1-7 J4 V3-15 J4 H4sH17730P V3-11 D1-7 J4V1-17 J2 H4sH17930N V3-64 D2-2 J6 V1-39 J5 H4sH17930N2 V3-64 D2-2 J6V1-39 J5 H4sH21051P V3-23 D7-27 J4 V1-39 J5 H4sH21054P V3-23 D1-7 J4V1-39 J5 H4sH21055P V3-11 D7-27 J2 V1-39 J2 H4sH21058P V3-20 D2-2 J5V1-39 J2 H4sH21064P V3-64 D6-6 J6 V1-39 J5 H4sH21073P V3-43 D6-19 J3V1-39 J2 H4sH21077P V3-23 D6-19 J3 V1-39 J2 H4sH21079P V3-15 D1-7 J4V1-39 J2 H4sH21080P V3-23 D1-7 J6 V2-28 J1 H4sH21083P V3-23 D1-7 J2V3-15 J5 H4sH21086P V3-33 D2-21 J6 V4-1 J5 H4sH21090P V3-23 D1-20 J4V3-15 J4 H4sH21091P V3-15 D6-19 J6 V1-17 J4 H4sH21093P V3-33 D3-3 J3V1-6 J2 H4sH21099P V3-9 D1-1 J6 V1-39 J5 H4sH21100P V3-9 D1-7 J3 V1-39J5 H4sH21103P V3-15 D1-7 J4 V1-39 J5 H4sH21104P V3-11 D3-10 J3 V1-39 J5

Example 4: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-HLA-A2:HPV16E7 MonospecificAntibodies

Binding affinities and kinetic constants of human anti-HLA-A2/HPV16E7antibodies were determined via real-time surface plasmon resonance (SPR;Biacore 4000 or Biacore T-200, GE Healthcare Life Sciences, Pittsburgh,Pa.) at 25° C. Antibodies were captured onto a CM5 Biacore sensorsurface (GE Healthcare Life Sciences) derivatized via amine couplingwith a monoclonal anti-human Fc antibody (GE, #BR-1008-39). Variousconcentrations of monomeric HLA-A2: HPV16E7 peptide complex containingeither the E7:11-19 peptide (SEQ ID NO: 538) or the E7:82-90 peptide(SEQ ID NO: 539) were injected over the anti-HLA-A2:HPV16E7 antibodycaptured surface at a flow rate of 50 μL/minute (Biacore T-200) or304/minute (Biacore 4000). Antibody-reagent association was monitoredfor 4-5 min. and the dissociation was monitored for 10 min. All bindingstudies were performed in HBS-ET buffer (0.01M HEPES pH 7.4, 0.15M NaCl,0.05% v/v Surfactant P20).

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by fitting the real-time sensorgrams to a 1:1 binding modelusing Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as:

${{K_{D}(M)} = \frac{kd}{ks}},{{{and}\mspace{14mu} t\; 1\text{/}2\mspace{14mu}\left( \min \right)} = {\frac{\ln(s)}{{sc} + {kd}}.}}$

Binding kinetic parameters for the monospecific anti-HLA-A2:HPV16E7antibodies to monomeric HLA-A2/HPV16E7 peptide complex are shown belowin Tables 5 and 6.

TABLE 5 Biacore binding affinities of anti-HLA- A2/HPV16E7 (11-19)antibodies at 25° C. HLA-A2:HPV16E7(11-19) Antibody ka (1/Ms) kd (1/s)KD (M) t½ (min) H4sH17670P 8.16E+04 1.43E−03 1.75E−08 8.1 H4sH17672P1.29E+05 8.19E−04 6.37E−09 14.1  H4sH17673P NB NB NB NB H4sH17675P5.99E+04 1.38E−03 2.31E−08 8.4 H4sH17680P NB NB NB NB H4sH17697P NB NBNB NB H4sH17707P NB NB NB NB H4sH17715P NB NB NB NB H4sH17726P NB NB NBNB H4sH17730P NB NB NB NB H4sH17363N 8.72E+04 1.54E−03 1.76E−08 7.5H4sH17364N 8.56E+04 1.57E−03 1.83E−08 7.4 H4sH17368N2 NB NB NB NBH4sH17368N3 NB NB NB NB H4sH17930N 7.84E+04 7.96E−04 1.02E−08 14.5 H4sH17930N2 8.28E+04 7.92E−04 9.57E−09 14.6  H4sH21051P NB NB NB NBH4sH21054P NB NB NB NB H4sH21055P NB NB NB NB H4sH21058P NB NB NB NBH4sH21064P 5.47E+04 7.91E−04 1.44E−08 14.6  H4sH21073P NB NB NB NBH4sH21077P NB NB NB NB H4sH21079P 3.74E+04 1.09E−02 2.90E−07 1.1H4sH21080P 1.79E+05 3.90E−02 2.18E−07 0.3 H4sH21083P NB NB NB NBH4sH21086P NB NB NB NB H4sH21090P NB NB NB NB H4sH21091P NB NB NB NBH4sH21093P NB NB NB NB H4sH21099P NB NB NB NB H4sH21100P NB NB NB NBH4sH21103P NB NB NB NB H4sH21104P NB NB NB NB *NB indicates that underexperimental conditions, HLA-A2:HPV16E7(11-19) peptide reagent did notbind to the captured anti-HLA-A2:HPV16E7 monoclonal antibody

TABLE 6 Biacore binding affinities of anti-HLA- A2/HPV16E7 (82-90)antibodies at 25° C. HLA-A2:HPV16E7(82-90) Antibody ka (1/Ms) kd (1/s)KD (M) t½ (min) H4sH17670P NB NB NB NB H4sH17672P NB NB NB NB H4sH17673PNB NB NB NB H4sH17675P NB NB NB NB H4sH17680P NB NB NB NB H4sH17697P NBNB NB NB H4sH17707P 7.15E+04 3.61E−04 5.05E−09 32.0 H4sH17715P 4.58E+045.68E−04 1.24E−08 20.3 H4sH17726P 5.17E+04 4.19E−04 8.10E−09 27.6H4sH17730P NB NB NB NB H4sH17363N NB NB NB NB H4sH17364N NB NB NB NBH4sH17368N2 8.31E+05 1.92E−03 2.30E−09  6.0 H4sH17368N3 7.12E+051.22E−03 1.71E−09  9.5 H4sH17930N NB NB NB NB H4sH17930N2 NB NB NB NBH4sH21051P 1.37E+04 3.31E−04 2.41E−08 34.9 H4sH21054P 1.98E+05 7.65E−043.86E−09 15.1 H4sH21055P 1.56E+05 1.21E−03 7.76E−09  9.6 H4sH21058P2.46E+05 2.60E−04 1.06E−09 44.5 H4sH21064P NB NB NB NB H4sH21073P5.77E+05 1.15E−04 2.00E−10 100.3  H4sH21077P NB NB NB NB H4sH21079P NBNB NB NB H4sH21080P NB NB NB NB H4sH21083P 5.38E+04 2.12E−04 3.94E−0954.5 H4sH21086P 6.97E+04 1.14E−03 1.63E−08 10.2 H4sH21090P 8.11E+041.91E−04 2.35E−09 60.6 H4sH21091P 1.74E+05 1.46E−04 8.42E−10 79.1H4sH21093P 1.18E+05 1.92E−03 1.63E−08  6.0 H4sH21099P 1.24E+05 9.79E−057.88E−10 118.0  H4sH21100P 2.90E+05 1.82E−04 6.26E−10 63.5 H4sH21103P8.35E+05 3.22E−03 3.86E−09  3.6 H4sH21104P 4.36E+04 2.15E−04 4.94E−0953.7 *NB indicates that under experimental conditions,HLA-A2:HPV16E7(82-90) peptide reagent did not bind to the capturedanti-HLA-A2:HPV16E7 monoclonal antibody

The data demonstrate that a majority of the anti-HLA-A2/HPV16E7antibodies of this invention selectively bound to soluble HLA-A2/HPV16E7peptide complex, some displaying sub-nanomolar affinity. Someantibodies, however, displayed no binding to the HLA-A2/HPV16E7 complex.

Example 5: Prediction of Potential Off-Target Peptides

Given a target 9-mer peptide-HLA-A2 complex, an associated potentialoff-target peptide is defined based on three criteria: A) the peptide isa 9-mer and is predicted to bind HLA-A2, B) the peptide is similar tothe target peptide based on sequence homology, and C) the peptide isderived from a gene that is expressed in essential, normal tissues.Therefore, to predict potential off-target peptides associated withYMLDLQPET (HPV16 E711-19; SEQ ID NO: 538) and LLMGTLGIV (HPV16 E782-90;SEQ ID NO:539) the following methodology was used (generally see,Dhanik, Ankur, et al. (2016) BMC Bioinformatics 17(1):286).

As a first step, canonical human protein sequences were downloaded fromthe UniprotKB database (version September 2014) (Magrane, Michele, andUniProt Consortium. Database 2011 (2011): bar009) and all 9-mers wereextracted. This resulted in 11,118,076 peptides from 20,014 proteinsequences.

Next, the binding affinities of the peptides with HLA-A2 were computedusing NetMHCstab webserver (version 1.0) (Jorgensen, Kasper W., et al.(2014) Immunology 141(1):18-26). Peptides with affinity value <500 nMwere predicted to bind HLA-A2, and the rest were discarded resulting inthe remaining 338,452 peptides.

The peptide sequences were then evaluated for sequence homology with thetarget peptide. For each peptide, its Degree of Similarity (DoS) wascalculated to the target peptide. The DoS value represents the number ofidentical amino acids at identical positions between the two peptides.Peptides with DoS value <6 were rejected resulting in the remaining 21peptides in the case of HLA-A2/HPV16E7:11-19 and 78 peptides in the caseof HLA-A2/HPV16E7:82-90.

The genes corresponding to the 21 peptides were checked for theirexpression in the essential, normal tissues. The evaluation for theexpression was done using the gene expression data derived from the GTEx(Gene Tissue Expression) and TCGA (The Cancer Genome Atlas) databases asprovided by OmicSoft (Hu, Jun, et al. Bioinformatics (2012)28(14):1933-1934). The data was available in RPKM (Reads Per KilobasePer Million) values from 497 TCGA adjacent normal samples (across 15essential tissue types), and 2,928 GTEx normal samples (across 22essential tissue types). Tissues other than the breast, cervix,fallopian tube, testis, uterus, and vagina, were considered essential. Agene was considered to be expressed in the essential, normal tissues ifthe maximum of the 95 percentile expression in each essential, normaltissue type in the GTEx and TOGA databases is >=0.5 RPKM. ForHLA-A2/HPV16E7:11-19 (YMLDLQPET), out of the 21 peptides, 10 peptideswere derived from genes that are expressed in the essential, normaltissues. For HLA-A2/HPV16E7:82-90 (LLMGTLGIV), out of the 78 peptides,49 peptides were derived from genes that are expressed in the essential,normal tissues.

The 10 peptides constitute the predicted off-targets associated with thetarget YMLDLQPET-HLA-A2 complex (Table 7). Out of the 49 potentialpeptides predicted to constitute likely off-targets associated with theLLMGTLGIV-HLA-A2 complex, 13 were picked at random for experimentalvalidation and are listed in Table 8.

TABLE 7 Predicted off-target peptides similar toHLA-A2/HPV16E7: 11-19 (YMLDLQPET; SEQ ID NO: 538) Predicted No.Peptide Sequence Peptide Name Gene IC50 (nM) 1YMLDLQKQL (SEQ ID NO: 546) SH3GLB1: 244-252 SH3GLB1 9.2 2KMLDKNPET (SEQ ID NO: 547) CAMKK1: 388-396 CAMKK1 107.9 3YMFDLLLET (SEQ ID NO: 548) USP47: 691-699 USP47 3.5 4YTLDLQLEA (SEQ ID NO: 549) CHPF: 463:471 CHPF 132.8 5MMLILQAET (SEQ ID NO: 550) PKD1: 2694-2702 PKD1 244.3 6LMLPLQPCT (SEQ ID NO: 551) NBR1: 357-365 NBR1 487.8 7YILDLLPDT (SEQ ID NO: 552) CBL: 83-91 CBL 145.9 8YMEDLQELT (SEQ ID NO: 553) PPP4R4: 20-28 PPP4R4 482.1 9GLLDLDPET (SEQ ID NO: 554) SBK3: 285-293 SBK3 91.6 10VMKDLLPET (SEQ ID NO: 555) FNDC3B: 921-929 FNDC3B 379.9

TABLE 8 Predicted off-target peptides similar toHLA-A2/HPV16E7: 82-90 (LLMGTLGIV; SEQ ID NO: 539) Predicted No.Peptide Sequence Peptide Name Gene IC50 (nM) 1LLMGTFLSV (SEQ ID NO: 556) VPREB3: 9-17 VPREB3 5.9 2LLGGTLERV (SEQ ID NO: 557) B4GALT2: 4-12 B4GALT2 93.6 3LLMGSNTIV (SEQ ID NO: 558) GCAT: 312-320 GCAT 13.2 4LLQATLDIV (SEQ ID NO: 559) CYP39A1: 246-254 CYP39A1 88.7 5LLLTFLGIV (SEQ ID NO: 560) ALDH3A2: 467-475 ALDH3A2 85.4 6LLAGTLAGV (SEQ ID NO: 561) CLCN4: 79-87 CLCN4 11.0 7LLQDTLGHV (SEQ ID NO: 562) ZHX2: 234-242 ZHX2 50.5 8LLLAVLGIV (SEQ ID NO: 563) GRM6: 590-598 GRM6 64.4 9LVMETLCIV (SEQ ID NO: 564) IPO9: 582-590 IPO9 18.8 10LLNETLGEV (SEQ ID NO: 565) IPO4: 163-171 IPO4 25.8 11KLMGHLGVV (SEQ ID NO: 566) SF3B1: 969-977 SF3B1 11.2 12LLMCYLYIV (SEQ ID NO: 567) DOCK11: 1282-1290 DOCK11 2.7 13LLNKVLGIV (SEQ ID NO: 568) Human CNOT1: 1962-1970 CNOT1 247.8

Example 6: T2 Peptide Pulsing to Determine HLA-A2/HPV16E7 M Specificity

To determine anti-HLA-A2/HPV16E7 monoclonal antibody specificity,peptide-pulsed T2 cells loaded with target or off-target peptides(identified in the previous Example) were used. Experiments were carriedout as follows: For the exogenous loading of HPV16E7 target oroff-target peptides, T2 cells were rinsed in AIM V® Medium and countedwith a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience).Approximately 6 million T2 cells per T-75 flask were cultured for 24hours at 26° C. in 9 mL of AIM V® Medium containing 10 μg of human b2mand 100 μg of HPV16E7 peptide or off-target peptide (Tables 6 and 7).Peptide-loaded T2 cells were washed once with PBS without Ca2+/Mg2+ andcounted. Approximately 10,000 cells per well of the peptide-loaded T2 oruntreated T2 in cell washing buffer were seeded into the 96-well carbonelectrode plates (MULTI-ARRAY high bind plate, MSD) and incubated for 1hour at 37° C. to allow cells to adhere to the plate. Nonspecificbinding sites were blocked using 2% BSA (w/v) in PBS for 1 hour at roomtemperature. To the plate-bound cells, solutions ofanti-HLA-A2/HPV16E7:11-19, anti-HLA-A2/HPV16E7:82-90 or control antibodyin serial dilutions ranging from 1.7 pM to 100 nM, as well as solutionswithout antibody were added. Plates were incubated for 1 hour at roomtemperature, then washed to remove the unbound antibody using anAquaMax2000 plate washer (MDS Analytical Technologies). Plate-boundantibodies were detected with SULFO-TAG™-conjugated goat polyclonalanti-human IgG antibody specific for the Fc gamma fragment (JacksonImmunoresearch, Meso Scale Discovery) for 1 hour at room temperature.After washes, the plates were developed with the Read Buffer (MSD)according to manufacturer's recommended procedure, and the luminescentsignals were recorded with a SECTOR Imager 600 (Meso Scale Discovery)instrument. The luminescence intensity, measured in relative light units(RLU), was recorded to indicate the binding intensity of each antibodyat the range of concentrations. The ratio of cell binding signal foreach anti-HLA-A2/HPV16E7 antibody compared to isotype control at 11 nM,is reported in Tables 8 and 9 and is an indication of specificity. At 11nM concentration most antibodies displayed minimal binding to T2untreated cells. Not all antibodies were tested with all correspondingrelated off-target peptides. Those not tested are marked as NT for “NotTested”. Antibodies with a binding ratio of greater than 15 are marked(+++), with a ratio equal to or less than 15 but greater than or equalto 10 are marked (++), with a ratio less than 10 but greater than orequal to 3 are marked (+) and antibodies with a binding ratio less than3 were classified as non-binders and denoted as (−). In addition, directbinding signals (in RLU) were analyzed as a function of the antibodyconcentration and data fitted to a sigmoidal (four-parameter logistic)dose-response model using GraphPad Prism™. The EC₅₀ values, defined asthe concentration of antibody at which 50% of the maximal binding signalon cells is detected, were determined, where possible, to indicatepotency of each antibody. EC₅₀ values for binding to cell-surfaceHLA-A2/HPV16E7:11-19 or HLA-A2/HPV16E7:82-90 only, are also reported inTables 9 and 10.

Ten of 13 anti-HLA-A2/HPV16E7:11-19 antibodies of the invention bind toT2 cell-surface HLA-A2/peptide complex. Seven of these 10 antibodies(H4sH17670P; H4sH17675P; H4sH17363N; H4sH17364N; H4sH17930N;H4sH17930N2; and H4sH21064P are specific for the HLA-A2/HPV16E7:11-19complex. Three antibodies (H4sH17672P, H4sH21079P, H4sH21080P) showeddisplayed higher potency with EC₅₀ values below 1.1 nM. Three antibodies(H4sH17673P, H4sH17680P, H4sH17697P) did not bind to T2 peptide loadedcells and are denoted with a (−) in the first column of Table 9.

The cell binding results on T2 cells loaded with HPV16E7:82-90 targetand predicted off-target peptides are summarized in Table 9. Sixteen of21 anti-HLA-A2/HPV16E7:82-90 mAbs of the invention bound T2 cell-surfaceHLA-A2/peptide complex. Only 2 mAbs from this group (H4sH17368N2,H4sH21086P) showed specificity to the HLA-A2/HPV16E7 82-90 complex. Fiveantibodies (H4sH17730P, H4sH21051P, H4sH21054P, H4sH21055P, H4sH21077P)did not bind to T2 peptide loaded cells and are denoted with a (−) inthe Table 10.

TABLE 9 Binding Specificity of Anti-HLA-A2/HPV16E7:11-19 MonoclonalAntibodies Cell Binding EC50 (M) T2 + peptide cell binding specificitycompared to irrelevant hIgG4s isotype control at 11 nM T2 + HPV16E7HPV16E7 SH3GLB1 CAMKK1 USP47 CHPF PKD1 NBR1 CBL PPP4R4 SBK3 T2 AbPID11-19 11-19 244-252 388-396 691-699 463:471 2694-2702 357-365 83-9120-28 285-293 untreated H4sH17670P 1.3E−09 ++ − − − − − − − − − −H4sH17672P 4.5E−10 ++ − − + − − − − − − − H4sH17673P − − − − − − − − − −− − H4sH17675P 1.1E−09 + − − − − − − − − − − H4sH17680P − − − − − − − −− − − − H4sH17697P − − − − − − − − − − − − H4sH17363N 2.1E−09 ++ − − − −− − − − − − H4sH17364N 2.0E−09 ++ − − − − − − − − − − H4sH17930N 3.1E−09++ − − − − − − − − − − H4sH17930N2 5.8E−09 ++ − − − − − − − − − −H4sH21064P 2.2E−09 +++ NT NT NT − NT − − NT NT − H4sH21079P 1.1E−09 +++NT NT NT + NT − + NT NT − H4sH21080P 2.2E−10 ++ NT NT NT + NT − + NT NT− Cell binding signal at 11 nM, RLU Isotype Ctrl. − 1029 976 772 11021077 1123 820 1104 1038 945 847

TABLE 10 Binding Specificity of Anti-HLA-A2/HPV16E7:82-90MonoclonalAntibodies Cell Binding EC50 (M) T2 + peptide cell binding specificitycompared to irrelevant hIgG4s isotype control at 11 nM T2 + HPV16E7HPV16E7 VPREB3 B4GALT2 GCAT CYP39A1 ALDH3A2 CLCN4 ZHX2 AbPID 82-90 82-909-17 4-12 312-320 246-254 467-475 79-87 234-242 H4sH17707P 2.3E−08 + − −− − − − + H4sH17715P 2.9E−10 ++ − − − − + − − H4sH17726P 4.1E−10 ++ +++++ − ++ + ++ + H4sH17730P − − − − − − − − − H4sH17368N2 7.5E−10 ++ − − −− − − − H4sH17368N3 1.8E−10 ++ − − − − − + − H4sH21051P − − − − NT NT NTNT − H4sH21054P − − − − NT NT NT NT − H4sH21055P − − − − NT NT NT NT −H4sH21077P − − − − NT NT NT NT − H4sH21086P 5.0E−10 +++ − − NT NT NT NT− H4sH21090P 5.6E−10 +++ − − NT NT NT NT +++ H4sH21091P 1.7E−10 +++ − −NT NT NT NT +++ H4sH21093P 3.0E−10 ++ − − NT NT NT NT + H4sH21058P1.9E−10 ++ − − − − − − − H4sH21073P 9.0E−11 ++ − − − − − − − H4sH21083P3.3E−10 ++ − − − − + − − H4sH21099P 8.7E−11 ++ − − − − + − − H4sH21100P9.6E−11 ++ − − − − − − − H4sH21103P 4.5E−11 ++ − − − − + − − H4sH21104P4.8E−10 ++ − − − + − − − Cell binding signal at 11 nM, RLU Isotype Ctrl.− 835 871 926 872 562 856 725 797 T2 + peptide cell binding specificitycompared to irrelevant hIgG4s isotype control at 11 nM GRM6 IPO9 IPO4SF3B1 DOCK11 CNOT1 T2 AbPID 590-598 582-590 163-171 969-977 1282-12901962-1970 Non-Pulsed H4sH17707P − − − − − − − H4sH17715P ++ − − + − + −H4sH17726P ++ + − + + + − H4sH17730P − − − − − − − H4sH17368N2 − − − − −− − H4sH17368N3 − − − − − − − H4sH21051P NT NT NT NT NT NT − H4sH21054PNT NT NT NT NT NT − H4sH21055P NT NT NT NT NT NT − H4sH21077P NT NT NTNT NT NT − H4sH21086P NT NT NT NT NT NT − H4sH21090P NT NT NT NT NT NT −H4sH21091P NT NT NT NT NT NT − H4sH21093P NT NT NT NT NT NT −H4sH21058P + − − + − − − H4sH21073P + − − − − − − H4sH21083P + − − ++− + − H4sH21099P + − − + − + − H4sH21100P + − − + − − − H4sH21103P +++ + +++ ++ ++ − H4sH21104P − − − + − − − Cell binding signal at 11 nM,RLU Isotype Ctrl. 807 768 1067 702 776 780 860

As shown in Tables 9 and 10, anti-HLA-A2:HPV16E7 antigen-bindingproteins of the invention bound with high specificity only to thespecific HPV peptide (SEQ ID No: 538 in Table 9, or to SEQ ID NO: 539 inTable 10) as presented by HLA-A2 and did not bind to any off-targetpeptides presented by HLA-A2.

Example 7: Binding Specificity Analysis Using Peptide Pulsed T2 Cells &FACS Analysis

Relative binding and specificity of HPV16E7 antibodies were accessed byflow cytometry on NIH3T3 cells expressing HLA-A2 complex presentingeither HPV 11-19 peptide (3T3/HLA.A2/hB2M/HPV16E7:11-19) or HPV 82-90peptide (3T3/HLA.A2/hB2M/HPV16E7 (82-90). NIH3T3 cells expressing HLAcomplex was generated by transfecting human HLA.A2 (accession numberP01892), human B2M (accession number NP_004039.1) and an ubiquitinpeptide cassette (Lévy F., et al. (1996) Proceedings of the NationalAcademy of Sciences of the United States of America 93(10):4907-4912;Valmori D, et al. (1999) Journal of Experimental Medicine189(6):895-906) comprising either amino acids 11-19 of HPV16E7 (SEQ IDNO: 538) or amino acids 82-90 (SEQ ID NO: 539) (accession numberAKI85233) using lipofectomine 2000 (Invitrogen, Cat #11668) followed byselection for at least 2 weeks in 1 μg/ml puromycin, 500 μg/ml G418, and100 μg/ml hygromycin. To stain, cells were harvested using celldissociation buffer (Millipore, Cat #S-004-C) and counted. Cells wereplated in staining buffer (PBS, without Calcium and Magnesium (Irving9240)+2% FBS (ATCC 30-2020) at a density of 200,000 cells per well in a96-well V-Bottom plate and stained with three-fold serial dilutions (1.7pM-100 nM) of primary antibodies for 30 min. at 4° C. Following primaryantibody incubation, cells were washed once in staining buffer, andstained with an Alexa-Flour 647 conjugated secondary antibody (JacksonImmunoResearch, Cat #109-606-170) at 10 μg/ml for 30 mins at 4° C. Cellswere then washed and fixed using a 50% solution of BD Cytofix (BD, Cat#554655) diluted in staining buffer. Samples were run and analyzed on anintellicyt iQue flow cytometer to calculate mean fluorescence intensity(MFI). MFI values were plotted in Graphpad Prism using a four-parameterlogistic equation over a 12-point response curve to calculate EC₅₀values. The secondary antibody alone (i.e. no primary antibody) for eachdose-response curve is also included in the analysis as a continuationof the three-fold serial dilution and is represented as the lowest dose.EC₅₀ values (M) and max fold binding (fold changed from highest dose tolowest does) are shown in Table 11. Several antibodies specificallybound to either the 3T3/HLA.A2/hB2M/HPV16E7:11-19 or the3T3/HLA.A2/hB2M/HPV16E7:82-90 cell line. EC₅₀ values ranged from 5-500nM and fold binding ranged from 1.0× to 43.8λ.

TABLE 11 FACS Binding of HPV16E7 antibodies 3T3/HLA.A2/hB2M/HPV16E73T3/HLA.A2/hB2M/HPV16E7 Abtibody (11-19) (82-90) HEK293 Designation EC₅₀Max Fold EC₅₀ Max Fold EC₅₀ Max Fold *H4sH17363N 1.40E−08 11.4 ND 1.7 ND1.3 *H4sH17364N 2.40E−08 11.4 2.91E−08 2.3 ND 1.2 H4sH17368N2 ND 1.81.94E−07 9.1 ND 1.8 H4sH17368N3 ND 1.1 3.26E−08 6.1 ND 1.7 *H4sH17670P2.37E−08 6.7 ND 1.5 ND 0.8 H4sH17672P 3.72E−08 10.2 ND 1.4 ND 1.6H4sH17673P ND 1.8 ND 1.3 ND 1.6 *H4sH17675P 1.27E−08 5.1 ND 1.3 ND 0.65H4sH17680P ND 1.5 ND 1.1 ND 1 H4sH17697P ND 1.2 ND 1.5 ND 1.1 H4sH17707PND 1.5 ND 1.8 ND 2 H4sH17715P ND 1.7 6.60E−06 6.7 3.47E−08 3.1H4sH17726P 5.20E−08 28.11 7.19E−08 43.8 ND 2.0 H4sH17730P ND 0.95.80E−08 2.9 ND 0.9 H4sH17930N 1.73E−08 13.5 6.62E−08 10.3 1.53E−07 4.3*H4sH17930N2 2.78E−08 11.4 7.48E−08 3 3.93E−08 3 H4sH21051P ND 1.2 ND2.0 ND 1.2 H4sH21054P ND 3.8 ND 4.9 3.51E−08 3.6 H4sH21055P ND 1.4 ND1.5 ND 1.9 H4sH21058P ND 1.3 1.01E−08 8.6 ND 0.9 *H4sH21064P 2.05E−0812.3 6.60E−08 2.2 ND 0.7 H4sH21073P ND 0.9 4.09E−08 6.5 ND 0.9H4sH21077P ND 2.0 ND 1.5 ND 1.6 H4sH21079P   4.02−08 26.6 5.40E−08 20.5ND 1.3 H4sH21080P 3.10E−08 11.7 2.11E−08 7.4 5.19E−08 4.4 H4sH21083P ND1.5 3.31E−09 8.5 ND 1.4 H4sH21086P 5.537E−07  14.6 3.49E−07 22 ND 1.3H4sH21090P ND 1.6 1.85E−09 6.25 ND 1.1 H4sH21091P ND 1.5 3.74E−10 5.6 ND1.6 H4sH21093P ND 1.9 2.95E−08 3.9 ND 1.4 H4sH21099P ND 1 1.19E−09 4.8ND 2 H4sH21100P 3.55E−08 4.4 1.19E−08 10.3 ND 0.7 H4sH21103P ND 1.69.20E−09 6.3 ND 1.5 H4sH21104P ND 1.6 5.481E−09  8.5 ND 1 Isotype CtrlND 1 ND 1 ND 1.2 Antibodies with (*) were run together in a separateexperiment ND = EC₅₀ Not Determined when max fold binding was less thanor equal to 2 fold

The specificity of six HPV16E7:11-19 antibodies was furthercharacterized by assessing binding to T2 (174 CEM.T2) cells pulsed withHPV16E7:11-19, HPV16E7:82-90 or predicted off-target peptides (Table 7).To pulse, T2 (174 CEM.T2) were re-suspended in AIM V medium at a densityof 1×106 cells/ml (Gibco. Cat #31035-025). Cells were pulsed by adding10 μg/ml hB2M (EMD Millipore Cat #475828) and 100 μg/ml of the indicatedpeptide. T2 cells were then incubated overnight at 26° C., washed instaining buffer and stained with the indicated antibodies at aconcentration of 10 μg/ml following the protocol described above. WIvalues were calculated and presented as fold change over unstainedcells. Relative binding of the six HPV16E7:11-19 antibodies on T2 cellspulsed with HPV16E7:11-19 range from 986-1200 fold above unstainedcells. No significant binding above isotype control was observed on T2cells pulsed with the other peptides (Table 12).

TABLE 12 FACS Binding of HPV16E7 antibodies to T2 pulsed cells. (Foldchange over unstained) HPV16E7:11-19 SH3GLB1 CAMKK1 USP47 CHPF PKD1 NBR1CBL PPP4R4 SBK3 Unpulsed YMLDLQPET 244-252 388-396 691-699 463:4712694-2702 357-365 83-91 20-28 285-293 Cells H4sH17363N 1001.4 13.6 2.00.6 7.4 1.2 3.6 19.5 6.1 0.4 8.5 H4sH17364N 986.1 15.2 1.0 1.3 10.9 1.03.0 23.9 9.0 0.8 11.7 H4sH17670P 1005.5 3.7 2.9 5.3 3.6 3.5 2.2 2.0 2.92.6 5.0 H4sH17675P 1204.2 10.5 2.4 13.3 5.0 2.9 2.2 4.9 5.5 2.9 7.2H4sH17930N2 1166.7 28.2 2.6 8.9 7.5 3.3 3.3 4.6 7.3 3.0 6.7 H4sH21064P1204.2 10.5 2.4 13.3 5.0 2.9 2.2 4.9 5.5 2.9 7.2 Isotype Ctrl 17.1 8.56.3 11.5 9.9 9.8 8.7 9.0 8.0 6.8 10.2 Secondary Alone 14.2 3.7 5.8 5.24.8 4.4 3.7 4.4 4.0 4.1 6.5 Unstained 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.01.0 1.0 1.0

Example 8: Epitope Analysis Using Alanine Scanning Peptides

Alanine scanning was performed to determine which residues in theHPV16E7:11-19 peptide were critical for antibody binding. T2 cells werepulsed with alanine scanning peptides and stained with HPV16E7:11-19antibodies as described above. The following alanine scanning peptideswere used (Table 13).

TABLE 13 Alanine scanning peptides used in the study SEQ ID NO: PeptideAla substitution 569 AMLDLQPET Y11A 570 YALDLQPET M12A 571 YMADLQPETL13A 572 YMLALQPET D14A 573 YMLDAQPET L15A 574 YMLDLAPET Q16A 575YMLDLQAET P17A 576 YMLDLQPAT E18A 577 YMLDLQPEA T19A

Conversion of aspartate 14 to alanine (D14A) and glutamine 16 to alanine(Q16A) drastically reduced antibody binding for all the testedantibodies. Conversion of tyrosine 11 to alanine (Y11A) reduced bindingof H4sH17670P, H4sH17675P, H4sH21064P, and H4sH17930N2; but notH4sH17363N or H4sH17364N. Conversion of leucine 13 to alanine (L13A) andproline 17 to alanine (P17A) reduced overall antibody binding (Table14).

To summarize, D14 and Q16 are critical residues for antibody binding.

TABLE 14 FACS Binding of HPV16E7 antibodies to T2 cells pulsed withalanine scanning peptides AMLDLQPET YALDLQPET YMADLQPET YMLALQPETYMLDAQPET (Y11A) (M12A) (L13A) (D14A) (L15A) H4sH17363N 694.8 998.5529.9 58.2 1019.3 H4sH17364N 708.3 997.9 489.9 69.7 1008.4 H4sH17670P8.1 670.3 374.9 10.7 1062.8 H4sH17675P 19.0 823.8 527.2 6.3 1153.1H4sH17930N2 39.0 972.9 665.7 5.8 1245.9 H4sH21064P 19.0 823.8 527.2 6.31153.1 Isotype Ctrl 14.4 10.9 8.4 9.9 8.1 Secondary Alone 9.7 6.9 4.96.4 6.3 Unstained 1.0 1.0 1.0 1.0 1.0 YMLDLAPET YMLDLQAET YMLDLQPATYMLDLQPEA (Q16A) (P17A) (E18A) (T19) H4sH17363N 13.3 401.8 775.7 1034.3H4sH17364N 13.2 384.8 756.2 1075.7 H4sH17670P 6.2 168.8 614.7 1150.1H4sH17675P 5.8 371.1 808.3 1165.0 H4sH17930N2 6.8 531.9 1035.4 1330.3H4sH21064P 5.8 371.1 808.3 1165.0 Isotype Ctrl 9.0 8.2 9.1 9.6 SecondaryAlone 5.5 4.6 4.5 6.5 Unstained 1.0 1.0 1.0 1.0

Example 9: Reformatting HLA-A2/HPV16E7 Antibodies into ScFv for Use inChimeric Antigen Receptors

Six HLA-A2/HPV16E7:11-19 antibodies (17363N, 17364N, 17670P, 17675P,17930N2 and 21064P) were reformatted into VL-VH single chain variablefragments (ScFv) and placed into a chimeric antigen receptor (CAR)construct that used a CD8α hinge and transmembrane domain, 4-1BBcostimulatory domain, and a CD3ζ stimulatory domain (SEQ ID NOs:540-545). The HLA-A2/HPV16E7:11-19 specific CARs were cloned into alentiviral expression vector (Lenti-X™ Bicistronic Expression System(Neo), Clontech Cat #632181) and lentiviral particles were generated viathe Lenti-X Packaging Single-Shot (VSV-G) system (Clontech Cat #631276)according to manufacturer protocols. Jurkat cells engineered to expressan NFAT-luciferase reporter (Jurkat/NFATLuc cl.3C7) were then transducedwith the 6 different CAR constructs using RetroNectin® Pre-coated Dishes(Clontech, Cat #T110a) according to manufacturer's protocols. Followingselection for at least 2 weeks in 500 μg/ml G418 (Gibco, Cat#11811-098), CAR-T cell lines were generated.

Activity of CAR-T lines was assessed in a CAR-T/Antigen Presenting Cell(APC) bioassay.

To perform the bioassay, 50,000 Jurkat/NFATLuc cl. 3C7 CAR-T cells wereadded to Thermo-Nunc 96-well white plates (Thermo Scientific, Cat#136101) in 50 μl of assay media (RPMI media with 10% FBS and 1% P/S/G)followed by the addition of a 3-fold serial dilution of APCs (150,000cells to 200 cells) in 50 μl of assay media. The following APCs wereutilized: CASKI (HLA-A2+/HPV16+), CASKI cells overexpressing a singlechain version of HLA-A2 presenting the 11-19 or 82-90 peptide, HEK293(HLA-A2+/HPV16−), or C33a (HLA-A2+/HPV16−). The cell mixture wasincubated in a 37° C., 5% C02, humidified incubator for 5 hours.NFAT-Luciferase activity was measured using Promega One-Glo (Cat #E6130)and a Perkin Elmer Envision plate reader. Relative luciferase units(RLU) were generated and plotted in Graphpad Prism using afour-parameter logistic equation over an 8-point response curve tocalculate EC50 values. The zero APC condition for each dose-responsecurve is also included in the analysis as a continuation of thethree-fold serial dilution and is represented as the lowest dose. Maxfold activation was determined by taking the ratio of the highest RLU onthe curve to the lowest. All six HLA-A2/HPV16E7:11-19 CAR-T cell lineswere activated by CASKI cells that overexpressed the HPV16E7:11-19peptide with max fold activations between 2.5-32.3 fold. No CAR-T celllines were activated by the APCs that overexpressed the HPV16E7:82-90peptide or HEK293 and C33a cells. Interestingly, one CAR-T cell line,that used the ScFv from antibody 17675P was activated by native CASKIcells with a fold activation of 4.1 and an EC50 of 68654 cells (Table15).

TABLE 15 Activation of HPV16E7 (11-19) CAR-T's in a CAR-T/APC BioassayJurkat/NFATLuc Chimeric Antigen Receptor Construct 17363N 17364N 17670P17675P 17930N2 21064P EC50 Fold EC50 Fold EC50 Fold EC50 Fold EC50 FoldEC50 Fold APC (cells) Activation (cells) Activation (cells) Activation(cells) Activation (cells) Activation (cells) Activation CASKI ND 0.9 ND1 ND 0.8 68654 4.1 ND 0.9 ND 1.2 CASKI 11-19 5108 2.5 6632 10.4 4145 8.59703 32.3 7885 16.8 7220 20.7 CASKI 82-90 ND 0.9 ND 1 ND 0.9 ND 1.5 ND0.7 ND 0.9 HEK293 ND 0.8 ND 0.9 ND 0.8 ND 0.7 ND 0.7 ND 0.8 C33a ND 0.7ND 1.2 ND 0.8 ND 0.6 ND 0.6 ND 0.4 ND = EC50 Not Determined when maxfold binding was less than or equal to 2-fold

Increasing the amount of HPV16E7:11-19 presented peptide HLA-A2 shouldresult in an increase in the activation of the HLA-HPV16E7:11-19 CARs.It has been reported that interferon gamma can increase antigenpresentation by MHC class 1 molecules though up-regulation of theproteasome (Fruh K. and Yang Y. (1999) Curr Opin Immunol. 11(1):76-81).Based on this observation, it was determined whether wildtype CASKIcells or HEK293 cells pre-treated with interferon gamma could result inincreased activation of the CAR-T cell lines. CASKI cells and HEK293cells were pretreated with 500 units/ml recombinant human IFN-γ(Peprotech Cat #300-02) for 48 hours and then used in the CAR-T/APCbioassay as described above (Table 16). IFNγ pretreated CASKI cellsactivate all 6 HPV16E7:11-19 CAR-T cell lines with a fold activationranging from 2.4-10.6

TABLE 16 Activation of HPV16E7 (11-19) CAR-T's in the presence of IFN-γJurkat/ CASKI CASKI + IFN-g HEK293 HEK293 + IFN-g NFATLuc EC₅₀ Max EC₅₀Max EC₅₀ Max EC₅₀ Max CART (cells) Fold (cells) Fold (cells) Fold(cells) Fold 17363N ND 1.0 51837 2.4 ND 1.0 ND 0.81 17364N ND 1.0 64405.6 ND 0.86 ND 0.75 17670P ND 1.0 51360 2.7 ND 0.77 ND 0.78 17675P 138441.77 64903 10.6 ND 0.81 ND 0.71 17930N2 ND 0.97 57186 8.1 ND 0.75 ND0.71 21064P ND 1.0 55863 8.97 ND 0.8 ND 0.7 ND = EC₅₀ Not Determinedwhen max fold binding was less than or equal to 2-fold

To further asses the specificity of the HPV16E7:11-19 CAR-T lines in theluciferase assay, we used T2 cells as the APC and pulsed with predictedoff-target peptides (Table 17). Briefly, T2 cells were pulsed with athree-fold serial dilution of the indicated peptides (1.7 μg/ml to 100ng/ml). Following pulsing, 50,000 CAR-T cells were added to Thermo-Nunc96 well white plates (Thermo Scientific, Cat #136101) in 50 μL of assaymedia. Then, 50,000 pulsed T2 cells were added to the plates in 50 μLassay media. The cell mixture was incubated in a 37° C., 5% C02,humidified incubator for 5 hours. NFAT-Luciferase activity wasdetermined using Promega One-Glo™ (Cat #E6130) and a Perkin ElmerEnvision plate reader. RLU were plotted in Graphpad Prism using afour-parameter logistic equation over a 12-point response curve tocalculate EC₅₀ values. The un-pulsed condition for each dose-responsecurve is also included in the analysis as a continuation of thethree-fold serial dilution and is represented as the lowest dose. Maxfold activation was determined as described previously. All CAR-T celllines were activated by T2 cells pulsed with the HPV16E7:11-19 peptide.The Jurkat/NFATLuc CART line utilizing the ScFv from antibody 17364N wasactivated non-specifically by T2 cells pulsed withEndophilin-B1(SH3GLB1:244-252), Chondroitin sulfate synthase 2(CHPF:463:471) and E3 ubiquitin-protein ligase CBL (CBL:83-91). Allother CAR-T cells lines had no significant activation with anyoff-target peptide.

TABLE 17 Max Fold Activation of HPV16E7 (11-19) CAR-T's against T2pulsed cells. Jurkat/NFATLuc Chimeric Antigen Receptor Construct 17363N17364N 17670P 17675P 17930N2 21064P Peptide Max Fold ActivationHPV16E7:11-19 6.5 10.9 1.9 10.6 7.8 12.9 HPV16E7:82-90 0.9 0.9 0.9 0.90.9 0.8 SH3GLB1:244-252 1.3 6.1 0.9 1.1 1.4 3.4 CAMKK1:388-396 0.9 1.00.9 0.9 0.9 0.8 USP47:691-699 1.0 0.9 0.9 1.0 0.9 1.1 CHPF:463:471 1.15.2 0.9 1.0 0.9 1.1 PKD1:2694-2702 0.8 0.9 1.0 1.0 0.9 0.9 NBR1:357-3650.9 0.9 0.9 0.9 0.9 0.9 CBL:83-91 1.3 7.2 0.9 1.1 0.9 1.5 PPP4R4:20-280.9 0.9 1.0 1.0 0.9 0.9 SBK3:285-293 1.0 0.9 1.0 1.0 0.9 0.9

Example 10: Structural Analysis of Fab Binding to HLA-A2+HPV16E7:11-19Peptide

In an effort to better understand the specific interactions betweenantibody and HLA-peptide complex, X-ray crystal structures of anantibody Fab fragment bound to HLA-A2/b2m displaying the HPV16E7:11-19peptide were determined. One structure contains the 17670P Fab, and theother structure contains the 17363N Fab; together, these two structurescover the sequence space of the six antibodies presented above (e.g.Table 11 and Table 12). All 9 residues of the HLA-displayedHPV16E7:11-19 peptide are clearly visible in the electron density mapsfor both 17670P and 17363N structures. Even at 2.9 Å (the resolution ofthe 17670P structure), the position and identity of the peptide residuesis unambiguous, and residue-residue interactions can be determinedaccurately. The 17363P structure is 2.6 Å, allowing improved accuracy.

The 17670P and 17363N Fabs bind to the top of the HLA-peptide complex,in a manner very similar to the way that TCR binds. The Fabs arepositioned and oriented almost identically to each other; both arealigned fairly parallel to the “rails” bordering the peptide bindinggroove, and both are centered on the bound peptide, with the heavy chainCDRs contacting the N terminal half of the bound peptide, and the lightchain CDRs contacting the peptide's C terminal half. Other publishedantibody complex structures (e.g. PDB codes 1W72 and 4WUU) reveal thatthe antibody does not have to cover the entire HLA-displayed peptide.However, these antibodies with only partial peptide coverage have poorspecificity, tolerating extensive changes in the part of the peptidethat is not contacted with little loss in binding affinity.

The structures show that the 17670P and 17363N Fab heavy chains contactresidues 11, 14, 15 in the HPV16E7 peptide, while the Fab light chainscontact residues 15, 17, 18. No Fab contacts are made with side chainsof residues 12, 13, 16, or 19 as they point toward the HLA molecule. Thebound peptide is numbered according to the residue positions in theoriginal HPV16 E7 protein, as follows:

(SEQ ID NO: 358) Y  M  L  D  L  Q  P  E  T  11 12 13 14 15 16 17 18 19

The majority of Fab contacts are made with the peptide side chains, notthe backbone.

Peptide contacts made by 17670P are concentrated almost exclusively inCDRs LCDR1 and HCDR3, particularly HCDR3. In particular, Fab heavy chainresidues 100, 101, 102, 105, 109, 110 of SEQ ID NO: 34 and light chainresidues 30, 31, 32, 50 of SEQ ID NO: 42 make contact with the boundpeptide, while Fab heavy chain residues 28, 31, 32, 100, 102, 104, 109,110, 113 of SEQ ID NO: 34 and light chain residues 31, 50, 52, 53, 54,55, 92 of SEQ ID NO: 42 contact the HLA. “Contact” here can involvedirect or water-mediated hydrogen bonds, charge-charge interactions, orhydrophobic/van der Waals interactions. For 17363N, Fab heavy chainresidues 102, 103, 108, 111, 112 of SEQ ID NO: 506 and light chainresidues 28, 30, 32, 50, 68 of SEQ ID NO: 514 contact the bound peptide,while Fab heavy chain residues 28, 32, 100, 102, 103, 107, 112 of SEQ IDNO: 506 and light chain residues 31, 49, 50, 51, 52, 53, 55, 92 of SEQID NO: 514 contact the HLA molecule.

Of the six anti-HLA-A2:HPV16E7:11-19 antibodies, 17675P is the mostsimilar to 17670P in the CDR sequences that determine peptide binding,with 21064P and 17930N2 also sharing a high degree of similarity in thepeptide-binding CDR regions. The key contacts between 17670P and theHLA-peptide complex are mostly conserved in 17675P, 21064P, and 17930N2,thus the binding mode of these antibodies is likely to be the same asthat of 17670P.

In contrast, CDR H3 of 17363N has a very different sequence compared toCDR H3 from 17670P, and this sequence difference translates into astructural difference of CDR H3, altering contacts with the HLA-peptidecomplex in this region. For example, heavy chain Tyr 100 in 17670Pcontacts Tyr 11 of the bound peptide. The equivalent residue in 17363Nis Tyr 102 (this antibody's CDR H3 is two residues longer) and thisresidue does not contact peptide Tyr 11. Instead, Tyr 102 has reorientedto make contacts with the HLA molecule nearby.

The lead antibody 17364N has a very similar sequence to 17363N, and isidentical in all residues contacting the HLA-peptide complex. Thisantibody should have a binding mode very similar to that of 17363N, andthus different from 17670P, 17675P, 17930N2, and 21064P.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method of treating a subject having anHPV16E7-associated disease or disorder, comprising administering to thesubject a therapeutically effective amount of an isolatedantigen-binding protein that binds specifically to a conformationalepitope of an HLA-A2 presented human papillomavirus (HPV) 16 E7 peptide(HPV16E7 peptide), wherein the conformational epitope comprises one ormore amino acids of SEQ ID NO: 537 selected from the group consisting ofY11, D14, L15, P17 and E18, wherein the antigen-binding proteincomprises three heavy chain complementarity determining regions (CDRs)(HCDR1, HCDR2 and HCDR3); and three light chain CDRs (LCDR1, LCDR2 andLCDR3), wherein (a) the HCDR1 domain comprises an amino acid sequencehaving at least 90% amino acid sequence identity to the entire aminoacid sequence of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148,164, 180, 196, 212, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364,380, 396, 412, 428, 444, 460, 476, 492, 508, and 524; (b) the HCDR2domain comprises an amino acid sequence having at least 90% amino acidsequence identity to the entire amino acid sequence of an amino acidsequence selected from the group consisting of SEQ ID NOs: 6, 22, 38,54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222, 238, 254, 270,286, 302, 318, 334, 350, 366, 382, 414, 430, 446, 462, 478, 494, 510,and 526; (c) the HCDR3 domain comprises an amino acid sequence having atleast 90% amino acid sequence identity to the entire amino acid sequenceof an amino acid sequence selected from the group consisting of SEQ IDNOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224,240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448,464, 480, 496, 512, and 528; (d) the LCDR1 domain comprises an aminoacid sequence having at least 90% amino acid sequence identity to theentire amino acid sequence of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140,156, 172, 188, 204, 204, 228, 244, 260, 276, 292, 308, 324, 340, 356,372, 388, 404, 420, 436, 452, 468, 484, 500, 516, and 532; (e) the LCDR2domain comprises an amino acid sequence having at least 90% amino acidsequence identity to the entire amino acid sequence of an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 30, 46,62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262, 278, 294,310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518,and 534; and (f) the LCDR3 domain comprises an amino acid sequencehaving at least 90% amino acid sequence identity to the entire aminoacid sequence of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160,176, 192, 208, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392,408, 424, 440, 456, 472, 488, 504, 520, and 536, thereby treating thesubject.
 2. The method of claim 1, wherein the HPV16E7-associateddisease or disorder is HPV-associated cancer.
 3. The method of claim 2,wherein the HPV-associated cancer is squamous cell carcinoma.
 4. Themethod of claim 3, wherein the HPV-associated cancer is cervical cancer,anogenital cancer, head and neck cancer, or oropharyngeal cancer.
 5. Themethod of claim 1, wherein the antigen-binding protein is administeredto the subject in combination with a second therapeutic agent.
 6. Themethod of claim 5, wherein the second therapeutic agent is selected fromthe group consisting of a PD-1 inhibitor, a CTLA-4 inhibitor, anantibody to a tumor specific antigen, an antibody to avirally-infected-cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, abispecific antibody against CD20 and CD3, a dietary supplement such asan antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxicagent, surgery, radiation, a NSAID, a corticosteroid, an anti-HPVvaccine, and any other therapy useful for ameliorating at least onesymptom associated with the disease or disorder.
 7. The method of claim1, wherein the antigen-binding protein is administered subcutaneously,intravenously, intradermally, intraperitoneally, orally, intramuscularlyor intracranially.
 8. The method of claim 1, wherein the antigen-bindingprotein is administered at a dose of about 0.1 mg/kg of body weight toabout 100 mg/kg of body weight of the subject.
 9. The method of claim 1,wherein the antigen-binding protein has a property selected from thegroup consisting of: (a) binds monomeric HLA-A2:HPV16E7 11-19 (SEQ IDNO:538) peptide with a binding dissociation equilibrium constant (K_(D))of less than about 20 nM as measured in a surface plasmon resonanceassay at 25° C.; (b) binds monomeric HLA-A2:HPV16E7 82-90 (SEQ IDNO:539) peptide with a binding dissociation equilibrium constant (K_(D))of less than about 25 nM as measured in a surface plasmon resonanceassay at 25° C.; (c) binds to HLA-A2:HPV16E7 11-19 (SEQ ID NO:538)peptide expressing cells with an EC₅₀ less than about 6 nM and do notbind to cells expressing predicted off-target peptides as determined byluminescence assay; (d) binds to HLA-A2:HPV16E7 82-90 (SEQ ID NO:539)peptide expressing cells with an EC50 less than about 1 nM and do notsubstantially bind to cells expressing predicted off-target peptides asdetermined by luminescence assay; (e) binds to HLA-A2:HPV16E7 11-19 (SEQID NO:538) peptide expressing cells with an EC₅₀ less than about 30 nMas determined by flow cytometry assay; and (f) binds to HLA-A2:HPV16E782-90 (SEQ ID NO:539) peptide expressing cells with an EC₅₀ less thanabout 75 nM as determined by flow cytometry assay.
 10. The method ofclaim 1, wherein the HPV16E7 peptide comprises the amino acid sequenceof YMLDLQPET (SEQ ID NO: 538).
 11. The method of claim 1, wherein (a)the HCDR1 domain comprises an amino acid sequence having at least 95%amino acid sequence identity to the entire amino acid sequence of anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 236,252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460,476, 492, 508, and 524; (b) the HCDR2 domain comprises an amino acidsequence having at least 95% amino acid sequence identity to the entireamino acid sequence of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150,166, 182, 198, 214, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366,382, 414, 430, 446, 462, 478, 494, 510, and 526; (c) the HCDR3 domaincomprises an amino acid sequence having at least 95% amino acid sequenceidentity to the entire amino acid sequence of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88,104, 120, 136, 152, 168, 184, 200, 216, 224, 240, 256, 272, 288, 304,320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, and528; (d) the LCDR1 domain comprises an amino acid sequence having atleast 95% amino acid sequence identity to the entire amino acid sequenceof an amino acid sequence selected from the group consisting of SEQ IDNOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 204,228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436,452, 468, 484, 500, 516, and 532; (e) the LCDR2 domain comprises anamino acid sequence having at least 95% amino acid sequence identity tothe entire amino acid sequence of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 230, 246, 262, 278, 294, 310, 326, 342, 358,374, 390, 406, 422, 438, 454, 470, 486, 502, 518, and 534; and (f) theLCDR3 domain comprises an amino acid sequence having at least 95% aminoacid sequence identity to the entire amino acid sequence of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 16, 32,48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248, 264, 280,296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504,520, and
 536. 12. The method of claim 11, wherein (a) the HCDR1 domaincomprises an amino acid sequence having at least 98% amino acid sequenceidentity to the entire amino acid sequence of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84,100, 116, 132, 148, 164, 180, 196, 212, 220, 236, 252, 268, 284, 300,316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508, and524; (b) the HCDR2 domain comprises an amino acid sequence having atleast 98% amino acid sequence identity to the entire amino acid sequenceof an amino acid sequence selected from the group consisting of SEQ IDNOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222,238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 414, 430, 446, 462,478, 494, 510, and 526; (c) the HCDR3 domain comprises an amino acidsequence having at least 98% amino acid sequence identity to the entireamino acid sequence of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152,168, 184, 200, 216, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368,384, 400, 416, 432, 448, 464, 480, 496, 512, and 528; (d) the LCDR1domain comprises an amino acid sequence having at least 98% amino acidsequence identity to the entire amino acid sequence of an amino acidsequence selected from the group consisting of SEQ ID NOs: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 204, 228, 244, 260, 276,292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500,516, and 532; (e) the LCDR2 domain comprises an amino acid sequencehaving at least 98% amino acid sequence identity to the entire aminoacid sequence of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,174, 190, 206, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390,406, 422, 438, 454, 470, 486, 502, 518, and 534; and (f) the LCDR3domain comprises an amino acid sequence having at least 98% amino acidsequence identity to the entire amino acid sequence of an amino acidsequence selected from the group consisting of SEQ ID NOs: 16, 32, 48,64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248, 264, 280, 296,312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520,and
 536. 13. The method of claim 12, wherein (a) the HCDR1 domaincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196,212, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412,428, 444, 460, 476, 492, 508, and 524; (b) the HCDR2 domain comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 6,22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222, 238,254, 270, 286, 302, 318, 334, 350, 366, 382, 414, 430, 446, 462, 478,494, 510, and 526; (c) the HCDR3 domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88,104, 120, 136, 152, 168, 184, 200, 216, 224, 240, 256, 272, 288, 304,320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, and528; (d) the LCDR1 domain comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124,140, 156, 172, 188, 204, 204, 228, 244, 260, 276, 292, 308, 324, 340,356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516, and 532; (e) theLCDR2 domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,174, 190, 206, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390,406, 422, 438, 454, 470, 486, 502, 518, and 534; and (f) the LCDR3domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:
 16. 32, 48, 64, 80, 96, 112, 128, 144, 160,176, 192, 208, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392,408, 424, 440, 456, 472, 488, 504, 520, and
 536. 14. The method of claim13, wherein the HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 comprise anamino acid sequence set selected from the group consisting of SEQ IDNOs: 4, 6, 8, 12, 14, and 16; 36, 38, 40, 44, 46, and 48; 84, 86, 88,92, 94, and 96; 196, 198, 200, 204, 206, and 208; 284, 286, 288, 292,294, and 296; and 508, 510, 512, 516, 518, and
 520. 15. The method ofclaim 13, comprising a heavy chain variable region (HCVR)/light chainvariable region (LCVR) (HCVR/LCVR) amino acid sequence pair, wherein theamino acid sequence of the HCVR and the amino acid sequence of the LCVRof each HCVR/LCVR pair are each independently at least 90% identical tothe amino acid sequences of an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290,298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418,426/434, 442/450, 458/466, 474/482, 490/498, 506/514, and 522/530. 16.The method of claim 15, wherein the amino acid sequence of the HCVR andthe amino acid sequence of the LCVR of each HCVR/LCVR pair are eachindependently at least 95% identical to the amino acid sequence of anHCVR/LCVR amino acid sequence pair selected from the group consisting ofSEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242,250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370,378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498,506/514, and 522/530.
 17. The method of claim 15, wherein the amino acidsequence of the HCVR and the amino acid sequence of the LCVR of eachHCVR/LCVR pair are selected from the group consisting of SEQ ID NOs:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258,266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370, 378/386,394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498, 506/514,and 522/530.
 18. The method of claim 17, comprising an HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:2/10, 34/42, 82/90, 194/202, 282/290, and 506/514.
 19. The method ofclaim 1, wherein the antigen-binding protein is a full-length antibody,a Fab, a Fab′, a (Fab′)2, an Fv, a single chain Fv (scFv), a T-bodyconstruct, or a CAR.
 20. The method of claim 19, wherein theantigen-binding protein is a single chain Fv (scFv).
 21. The method ofclaim 1, wherein the antigen-binding protein is a human monoclonalantibody, or antigen-binding fragment thereof.
 22. The method of claim1, wherein the isolated antigen-binding protein that binds toHLA-A2:HPV16E7 is present in a pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent.
 23. A method of treatinga subject having an HPV16E7-associated disease or disorder, comprisingadministering to the subject a therapeutically effective amount of anisolated antigen-binding protein that binds specifically to aconformational epitope of an HLA-A2 presented human papillomavirus (HPV)16 E7 peptide (HPV16E7 peptide), wherein the isolated antigen-bindingprotein binds to the same epitope as an antigen-binding proteincomprising a heavy chain variable region (HCVR)/light chain variableregion (LCVR) (HCVR/LCVR) amino acid sequence pair, wherein the aminoacid sequence of the HCVR and the amino acid sequence of the LCVR ofeach HCVR/LCVR pair are each independently at least 90% identical to theamino acid sequences of an HCVR/LCVR amino acid sequence pair selectedfrom the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58,66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186,194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/306,314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418, 426/434,442/450, 458/466, 474/482, 490/498, 506/514, and 522/530, therebytreating the subject.